So, today, we're going to be talking about two different technologies, IPS reprogramming technology, and genome editing technology, and how we think that these are going to lead to better treatments for disease. Now, my own focus, as Zara mentioned, is on Alzheimer's disease, so a lot of this talk will be focused through that lens. But on the other hand, the technologies I'm talking about can be applied to a wide range of diseases as well. A little background on Alzheimer's disease, probably it's familiar to at least some people in this room. This is something that's affected my own family, my wife's family, perhaps some people in here. It's a leading cause of dementia worldwide. So, all Alzheimer's is dementia but not all dementia is Alzheimer's, it's just the most common form. Dementia comes from Latin, without mind. So, you have to have multiple cognitive functions failing to be considered having dementia. One out of 85 people worldwide is predicted to have Alzheimer's by 2050, because do you know with the greatest risk factor for Alzheimer's is? Aging. So, essentially, as we are aging better which is great more of us are getting older. We live much longer lives. It's also raising the amount of people who have Alzheimer's simply because you're living longer. And unfortunately, as probably people know, there is no cure or real treatment for Alzheimer's that really reverses the pathology. Now there are a few classes of drugs that can reduce symptoms in some patients for a certain amount of time and that can be beneficial for a limited amount of time, but it doesn't work in everyone, and eventually, they're going to fail. So, you can imagine this already is a huge social and economic burden and this is only going to get incredibly worse as time passes. Now, there's two kind of large buckets people put the disease in. The first in more rare form it's so-called early onset or Familial Alzheimer's disease. So, this is only two or three percent of cases. But these are the pure genetic forms of the disease. So, if you have the mutation for most of these, you have a hundred percent chance of getting the disease. You only need to have one bad copy, it's autism will dominate, you get the disease 30, 40, 50 as young as 18. But again these are, if you want to say the more severe forms of Alzheimer's. What most people get is late onset Alzheimer's, which is defined as age 65 and above. And there's no one genetic factor that will give you Alzheimer's. There are some very strong risk factors and some more minor genetic risk factors, but they're only increasing your susceptibility and some of this is interacting with the environment, such as age. So, there are two pathologies that are associated with Alzheimer's if you look at Alzheimer's patient brains, and essentially, these are aggregated proteins. One that's on the outside of the cell and one that is on the inside of the cells in the neuronal processes. So, the one on the outside is the so-called ABeta plaques that you may have heard of, so big clumps of this sticky ABeta peptide. Now, interestingly the plaques themselves do not correlate with the severity of disease. So, if you have more plaques, you don't necessarily have worse Alzheimer's. And part of that is thought it's these smaller aggregates that form before the plaques that may be the more toxic species. So, the way this works, is you have amyloid precursor protein which is cleaved by a couple of enzymes, we'll talk about in a minute. And this releases a beta or beta amyloid peptide as it is put here. This little peptide is very sticky and binds with each others that form oligomers, that are toxic themselves and eventually, also into these plaques. So, again, there is another protein inside the cell that it also is affecting disease. So, this is primarily the Tau protein, which is modified, it's phosphorylated as well as other modifications which again, cause it to get sticky with each other and then cause aggregates which build and cause Neurofibrillary tangles inside the processes. This can block things transporting up and down the neuron, as well as other things that can happen. And ultimately, this is probably mostly what's killing the cells, at least in my opinion. So, in terms of these two proteins, usually, there's a hierarchy thought of, if you manipulate ABeta in either animal models or in cells, you will drive Tau pathology, whereas if you manipulate Tau, you don't seem to affect ABeta. So, there seems to be this kind of link where ABeta leads to Tau, ultimately leads to disease progression. So, this is a very complicated side, we're not going to go over all of it but just all of the genetics so far for early onset Alzheimer's point to aberrant APP processing. So, APP is amyloid precursor protein which is essentially the substrate that is cut to make this ABeta fragment. So, APP can be processed in two different ways. So, this bottom way, it's cleaved a couple of different ways and it forms ABeta which is toxic. The other way it can be cleaved forms either benign products or potentially even beneficial products. So, there is mutations that cause early onset Alzheimer's and APP itself, including the rare Swedish mutation that I'll talk about later. Most of early onset mutations are mutations that affect this gamma secretase complex that also affect its ability to make ABeta. The other point of this is, all the parts of this pathway are essentially your major drug targets and the largest focus of Alzheimer's pharmaceutical research, where you can target trying to eliminate ABeta through antibodies or other methods, or chemical inhibitors of these pathways, or indirect activation of this pathway. And unfortunately, in over 100 clinical trials these have all failed. So, the question is, why are they failing? So, one hypothesis, which I think has some weigh in it, is that ABeta is actually building up a decade or two before you have symptoms. And that these strategies might work but you need to go in before you had the disease for them to be effective. We'll know the answer to that potentially because there is a clinical trial right now in the country of Colombia, where due to a founder mutation there's something like five thousand affected people with early onset Alzheimer's and they know approximately when they're going to get it. So, they are going in preemptively to target ABbeta before they have symptoms. So, if that works, that might say that some of these treatments we already developed might work, you just have to build to find good biomarkers and go in much earlier into the disease. The other possibility is that, which I also believe, is that we don't fully understand the disease and that our model systems while they've been informative, are not informative enough. And a strong way to complement this which I think is important is having a human specific system. And we know that some of these genes that are involved in this processing that the human version of the gene behaves differently than the mouse version of the gene which is usually where these diseases are modeled. So, I think that having human stem cell models of disease is a very important complement to really try to understand this better and hopefully, get a treatment. So, one way you can approach this is IPS cells or Induced Pluripotent Stem cells. So, as we talked about briefly earlier today this is taking adult cells and turning them into human embryonic stem cell like cells called IPSC or IPS cells. Again, this was a Nobel Prize in 2012, as well as for some cloning experiments which is technology that led to, essentially, IPS cells. In the hypothesis of Dr. Yamanaka, was that if you added transcription factors and again transcription factors or genes that turn on the expression of many, many other genes. So, you could view them almost as master genes. By adding them, versions of these that are important for human embryonic stem cell identity, that are important for those cells to be what they are, that you could revert some adult cells back to this embryonic stem cell like state. From my perspective, this is a pretty gutsy experiment. It would be a little hard to get funded. But, like most things in science or probably everything in science, there is work before that that new discoveries are built on. So, there was lines of evidence that would say that this was possible. So, cloning technology. The idea here is that we can take an adult cell and turn it back to an embryonic stem cell, like cell to clone the animal. Now, this had not been done successfully in humans when Dr. Yamanaka made IPS cells. It has since been done, but at the time, it had not been done but we knew we could do this in other mammals. So the other bit of evidence is that we know if we add transcription factors, that that could change cell fate. And what I mean by that is, that you can take one cell type, add a transcription factor and then it can become another cell type. So in this particular experiment, they could take fibroblast skin cells, add a single transcription factor MyoD and that led to the production of myoblaster skeletal muscle. So just this one transcription factor was enough to change it. So, this kind of evidence before that gave strength that this kind of idea would work. But how does this work? How do you make iPS cells? So the first thing you need to do is isolate adult dividing cells, cells have to be dividing. This division is part of the reprogramming process. So, if you take a terminally differentiated cell, such as a nerve cell in your brain, that can't undergo cell cycle, you cannot reprogram it to an iPS cell, it has to be able to divide. Now, typically people use skin and blood. The reason people use skin and blood is that it's easy to get from subjects. In theory, any dividing cell in your body can be reprogrammed and many different kinds have been reprogrammed. But these are particularly accessible and easy to get from people. Now, just to show how far this technology can be pushed, although, I don't necessarily think a lot of people can do this. Apparently, there is enough viable adult cells in your urine to be able to reprogram those into stem cells. So, in the case of skin, you isolate fibroblasts similar to what we just saw. In the case of blood, you isolate peripheral blood mononuclear cells and you can isolate renal cells from urine. Just thinking about the time scale, this takes like a couple of weeks to over a month, to be able to get these adult cells in enough levels to reprogram. The next step is you add these stem cell factors. So in Dr. Yamanaka's original paper, he added 24 factors initially, and then through trial and error basically whittled it down to these four, and these four are the same that I use in my own lab and what most people use, although there are other versions as well. So this OCT4, KLF4, SOX2 and c-MYC are four different transcription factors that when you put them in, and they only need to express for a limited amount of time, it will turn a minority of adult cells into a human embryonic stem-cell-like-cell. Now you can bring these in by a variety of means. So, the first versions of this was with integrating virus, meant that the virus put it inside the genome of the cells, which you can imagine you could interrupt in a lot of places. So that was kind of a recipe for cancer out of these cells. But the other versions, excuse me, use non integrating virus or DNA and RNA or ways that are not going to change the genome of the cells. So that would be obviously a lot safer for a cell transplant. You add them transiently in and three black-box process, although it's partially understood, all the genes that are important for your fibroblast identity, your blood identity start to shut off. In all your genes that are important for your stem cell identity including these four, which were sitting there dormant in the cells turn on and then you get this iPS cell that you can more or less divide indefinitely and use the disease model or use for transplants at some point as well. And this takes, if everything is working nicely, two or three months to do. So you're talking the whole process, best case scenario two months to four months if everything is moving very smoothly. In which case then you can work with these cells. So, of course, once you have the cells you want to make them into different cell types unless you want to study stem cells themselves. And people do do that. So, I'm a neuron guy, I'm a neurobiologist, as well as a stem cell biologist so I love neurons and our nerve cells, but I will fully admit that in a dish by far the cool cells are cardiomyocytes or heart cells when you see these. Because these cells will electrically communicate with each other and not only that, they will communicate and synchronize and beat together like your own actual heart. So, the first time you see this under a microscope, it's pretty amazing. But this is a video taken by my colleague and friend Dario at Columbia University, who is part of the Columbia University's Stem Cell Core. He makes boatloads of these cells all the time. As we look in the video, they just beat the dish by themselves at their own rhythm. And you can use this both to study cardiac diseases, so you can make stem cells from people who have certain risk factors that give them early heart attacks, or also people are developing trials to basically make a stem cell derived cardiac patch for regenerative medicine. So, cardiac cells are used in both of those contexts. So now we have this idea that we want to make iPS cells to study Alzheimer's, but how would we do this? What are the kinds of cells we would start with? And there's different options you could do. So, what I first did and this is when I was at New York Stem Cell Foundation, what our lab did, as well as other labs, is started with the pure genetic forms of the disease, the early onset form of Alzheimer's. In these cases you definitely know the mutation is sufficient to cause the disease and that you can be sure that you know what you're talking about. So, as I mentioned these are Izumo dominant mutations so this is a fictitious pedigree, but in a pedigree your boxes are your males, your circles are your females, in this pedigree the red are affected, and you can see roughly half of your offspring will get the disease in these families. So we could make stem cells from both unaffected and affected members of the same family, that would share genetics, but not have the early onset Alzheimer's mutation. So, we did this by using fibroblast or actually collect in the mid 1980s, from two different families the so-called FAD1 and FAD4 families, otherwise known as the Canadian and Italian families. So these were two of the four original families that identified PSEN1 is sufficient to cause early onset Alzheimer's disease. So, PSEN1 is part of this gamma secretase complex that I talked about a minute ago, which is involved in the processing of a beta as well as other functions. So, what we did is we made them into neural progenitors primarily, as well as neurons. We chose to look at progenitors, because we thought it might be a more homogeneous population and we were able to demonstrate as expected, that the mutant cells would make more of the sticky form of a beta. So the longer forms of a beta are more sticky and soft as mutations in PSEN1 create more of the sticky form, and that's what our cell showed as well. So while I think it's, important to study these strong mutations, on the other hand, that's not what most people get. Most people have late onset. So we also want to be able to model late onset Alzheimer's. Now, there's different ways you can do it and again this is at New York Stem Cell Foundation when we did this. Our idea was, let's make them from Alzheimer's patients, but we need to know that they definitely have Alzheimer's. So although imaging studies are getting better and better, and can now predict at a pretty high level, you cannot tell definitively somebody has Alzheimer's until a post-mortem autopsy. There is other dementias that clinically present very similar, but don't have the same pathology and probably have very different underlying genetics. So if you made stem cells from those persons it may be very confusing or add a lot of other extra information. So, this idea was not mine. This was a neuropathologist we were collaborating with at the time at Columbia. So, kind of off the cuff, suggested that we make cells from the New York brain bank, so we thought we would do this and this would allow definitive Alzheimer's diagnosis so we could rule out other dementias, and you could also potentially stratify subjects based on disease pathology. So you could look at their brains since there was an autopsy, you can know if they had mild, severe, or moderate a beta pathology, Tau and you could kind of try to tease out these differences. So, I told my boss at the time, I want to do this and he kind of smirked at me and said, "Go ahead". Supportive but skeptical. And this is because these cells were never frozen with the intent of growing out cells. Now they were frozen carefully in the sense that they went to maintain the architecture of the brain, but normally say I was going to freeze down my cells that I'm studying my stem cells, you add an antifreeze to them. Similar to what you do with your gasoline. So you add in this case DMSO, so when you take your cells and freeze and thaw them you don't form ice crystals which can puncture and essentially kill a lot of your cells. So the idea is, they're going to be anything alive left in these brains that were more or less flash frozen. So, what I did, which is the first step that many scientists do, is you look to see what's in the literature? You look to see what people did before you? Is there any evidence out there that suggests this may work? So it turns out there was one paper from 2009, by a Japanese group, where they took a bull testicle in particular Yasufuku's bull testicles, so this apparently was one of the founding cattles of Wagyu beef, which Kobe beef is a derivative of. So they had this testicle in the freezer and they wanted to see if they could clone the cow, essentially. So, they were able to grow fibroblast-like cells out of this frozen tissue and then they could actually clone the cow, that was again in 2009. So again, this is a cow but it's the idea that you can take tissue that was frozen, a very suboptimal way, grow some kind of cell that's viable and reprogram it in some fashion. So with that after pilot experiments, we actually took a bank to Alzheimer's brain as well as other diseases. So again, we have the pathology so we know it has a strong a beta load and also modify Tau. It was 10 years in the freezer, and we took actually not the brain itself but the dura matter which is above the brain beneath the skull, and we were able to grow fibroblasts-like cells out of there. It took a month. So basically the team I recruited, just kind of fed the cells and looked at every couple of days, and eventually these cells started crawling out. They literally, when you do this, or if you do a skin punch from a fresh patient, literally the cells will start to crawl out of the tissue. Yes a little freaky. We were able to reprogram at least, some of these to iPS cells and then we could use those to differentiate them into cortical neurons, which are vulnerable in Alzheimer's disease. So for my current work while we're interested in neurons we're also interested in other cell types that may also be affected in the disease. So one of the strength of stem cells right is they can make all these different cells. So once I have these lines I don't just have to make neurons. I can make in this case brain endothelial cells or vascular cells which are also damaged in the disease. You can make astrocytes, support cells which also may be affecting the disease. So you have this ability to model many, many different things with one starting material. So this is in collaboration with Dr. Drewton Agglieau at Columbia and what we do, this is kind of unusual differentiation protocol. So most differentiations you kind of mimic development, at least most protocols. So you look what was known in mouse and other systems and you add certain developmental pathways to push to one level of tissue and then another level of tissue as we saw earlier and then you go specifically to your tissue of interest and you try to mimic that. Here are what we do for the first six days or so is we put in media that does not support pluripotency. So they will not remain stem cells but we don't direct them towards anything in particular. So they can just make whatever they want. In the cell for part of what they make is neurons or brain cells and part of what they make is vascular cells and then you slam them into conditions that support vascular cells and propagate and expand those cells. So there is a variety of things you can do to test to make sure you're making what you think you are. So for instance, so this vasculature is part of the blood brain barrier, it's not the only cell, part of the blood brain barrier, astrocytes, parasites other things contribute. But it's part of this layer that protects your brain from incoming toxins. It also makes it challenging for drugs to get through when you want to do treatments too. So it's a plus or minus. So, you know we can look for markers that are at these junctions that are important for maintaining this blood brain barrier. We can do tests like a dye to see how permeable it is, the dye to go from one side to the other et cetera. And we can make these human brain endothelial cells. Now, the tricky part of this though is that although this IMR90 cell one which we actually have here is exceptional in making these brain endothelial cells. That is not true for every stem, so we try to do this with or in the original study that was published. And this is probably due to the undirected component of this protocol. So if you let some cells go wild as I like to say, different stem cell lines will go in different direction by chance or by their genetics and they have a tendency to consistently go in that direction. So some cells happen to consistently make neurons very well. So in this case vasculature comes from the mesadieuerm which of the three germ layers is kind of the most complicated to get because you need crosstalk from the other two. So I don't think a lot of cell lines necessarily spontaneously make a lot of mesadieuerm. So this is challenging in some ways, right? So you have this works great in the cell line but it's not necessarily going to work for all the other lines you model. And the question is how do we get around this problem? Now, one way to get around this problem is just a change of protocol although there isn't another one but one could try to develop one. Another way to do it is what I'm going to talk about next for the remaining part of my talk which is genome editing, well also known as gene editing where instead of taking Alzheimer's patients cells I can make control cells Alzheimer's cells by adding mutations to them using this technology. And by editing IMR 90 which we've done successfully and adding on early onset genes we can then early onset Alzheimer's Racine's, we can model these vascular cells with Alzheimer's and non-Alzheimer's cells. So, what is this? We are changing the genome at specific locations for instance the disease relevant gene. You can be used to add or remove mutation. So I just talked about adding it to disease model but you can imagine if you wanted to do stem cell replacement and you have some mutation that's making your cells sick or not function properly. That if you were going to take your cells and try to replace them you might want to correct that mutation. So you can also remove that mutation in the IPS cells you generated from a patient and then put it back in their body in such a way that they're not going to get sick again. So what we are trying to do is change one or a handful of base pairs out of the 3 billion that are in the human genome. So this seems pretty daunting. How do we do this? So there's at least three technologies that enable you to do this but what we're showing here which is CRISPR Cas 9 the third of these by far has revolutionized the field compared to the other two technologies. It's made it far more accessible for many, many labs to do this although I wouldn't say it's trivial but it opens up the doors for many people. So this is an example of basic science technology that people are just studying bacteria that you can use this knowledge in ways that are revolutionizing scientific discovery. So I am very supportive of a very broad scientific funding because you don't know how one, it's important to understand how the world works but also you don't know how this is going to impact your other research. So, how does this work on a technical level? So you have part of this which is an RNA. This CRISPR RNA that binds the genome. So the genome remember is a series of base pairs HTG and C, that's the genetic code, sets of three of them make code for an amino acid. So essentially you have 20 different base pairs that can all be HTGC and you can view that almost like your zip code in the genome. So it's going to bind one specific location in the genome. In this case say an early onset Alzheimer's risk gene. So we'll bind this specific location. So that's the CRISPR part. There's kind of a trans activating adapter that binds this Cas 9 protein and essentially what the Cas 9 protein is acting as molecular scissors. So it is chopping both strands of the DNA at that location. Now you may say, "Why would you want to chop the DNA at that location?" That sounds like an imitation for cancer for instance. So if you had too much UV light for instance you can get double strand breaks from that and that can lead to cancer cells. The reason that works is when you have double strand breaks the DNA needs to be repaired and the cell repairs it. If you don't add in anything additional to the cells often that repair is faulty. So that can lead to loss of gene function. So if that happens in an oncogene in your normal cells that can help lead the cancer. But in this case you can also use that to interrupt gene function and that's how we can knock out a gene or remove its function. But mostly what I'm interested in doing is adding mutations or removing mutations. So in that case we add a template. So when this is repairing, this template that I add additionally can be inserted in any changes that may cure can be added to the genome. Now that correct repair is a fairly rare event. The best I've gotten is roughly 8 percent of the cells that I'm screening. Typically it's more like 1% to 5%. So you end up screening many, many individual lines, sometimes a couple of hundred individual lines up each one in their own little well. So this is a fairly labor intensive process but it does work and we can add these mutations. So what we're doing now is we are creating cellular libraries of mutations. And what I mean by that is taking one cell line and adding early onset mutations that are sufficient to cause disease as well as late onset mutations that are risk factors all in the same genetic background so we can really start to cross compare all these risk factors essentially in the same cell line. So you can imagine if we made stem cells from everyone in here they would be very different from each other. We're all very different. There's a lot of noise even true in cell lines you made from early onset mutations. There are still a lot of noise in the system because people are different. So by having this called iceogenics system where cell lines are exactly the same except for the mutation, you reduce a lot of this noise. And you can do this using either human embryonic stem cells or IPS cells as a background to add these mutations, I've done both. So, here's an example of that. So, this is knockin the APP Swedish Mutation that I mentioned so this mutation is quite rare. It's actually I think all in one family in Sweden. But it is very commonly used in animal models into the H9 human embryonic stem cell line. So, if you look here, you have A G A T G so that codes for lysine and thianine. So, this is in control cells and then, we can just simply change it to A T C T A and then you get a spare gene in leucine which is the actual amino acids that are in the Zs and in theory, every other base pairs is the same between these. Anytime you bring cells to a clonal level, you are just dealing one cell line. You could have random changes introduced but more or less cell line you have produced from these, are going to be exactly the same except for this mutation. And then, you can make them into nerve cells including expression of tauwhich is this protein that aggregates on the inside of cells during Alzheimer's. Now, you can grow cells in a monolayer but there's advantages and disadvantages of that. And advantage is for drug screen, it's great. If you have flat cells that you can look at and you can image very easily. So, I think that's very useful. But again, that's not how our brain our brain is not flat. Our brain is 3D and has a lot of cells in interacting so you want to be able to try to get closer to that too. So, we're also trying to do that using so-called Cortical Spheroid. There are different types of kind of mini brain protocols. Often it's called Organoids or Cerebral Organoids. This particular protocol is called cortical steroids and these are 3D floating mini brains to get to a couple of millimetres big that you have both cortical neurons and astrocytes. Now, does it have everything in your brain? No. It doesn't have a good dendrocytes which do myelinization. It doesn't have vascular cells. Also, it doesn't have microglia which are the immune cells of your brain. But it's a lot closer, at least because it's 3D and it at least has multiple cell types and people are trying to build better and better 3D many brains to get even closer to a real brain. So, the hypothesis was that A beta would build up in these cells and cause pathology. So, we could show we can make more A beta in kind of these flat cells in a dish. We can imagine, we are constantly taking media on and off and that's going to wash it away because the A beta is on the outside of cells. But here we have steroids where things can build up on the inside. And on top of it, unlike a normal brain, you don't have vasculature to clear that A beta away. You don't have microglia there, immune cells that can eat it. So, that's. It is a pretty long experiment that was four and a half months, which in the so-called world is pretty long. So, if you look at our control cells, these little blue dots, every little blue dot here is a cell. So, if you look at brown that is an antibody against A beta in what we start to see only in our cells that have the APP Swedish mutation knocked in is we see these little extra cell aggregates. Now, I talk to a neuropathologist about this. They don't look exactly like plaques in the human brain. They are too symmetrical. But on the other hand, this doesn't have a lot of other cell types and this is still an artificial system, but there are at least, somewhat reminiscent of amyloid plaques. So, we kind of set up a system, if we want to address, how this affects the function of the cells that are toxic. How does it affect their electrical activity. So, some of the earliest effects in Alzheimer's is in cells dying. Eventually, huge parts of your brain do die. But early on a lot of the early issues are more cells communicating with each other. Synoptic problems. So, we want to look at do these cells have deficits in that? So, what I've showed you so far is essentially we can replicate parts of the disease in a dish. Now, that's important because it validates the system and also it's important for drug discovery efforts if you want to go after some of these pathologies. But on the other hand, I think these cells are going to be the strongest when we learn new things about the disease and understand it better than we understood before. So, there's a lot of remaining questions in Alzheimer's. These are just three of them but three major ones, at least major ones that are important to me. So, the first one is late-onset Alzheimer's one disease or a spectrum disorder similar to autism where it's a bunch of closely related diseases that are going to have some overlap and some differences. My personal opinion it is actually a spectrum disease. Some people do feel that way. It isn't necessarily universally accepted. And that one can imagine if people have different aspects, you might treat people clinically different. And this comes to this whole idea of personalized medicine or precision medicine as it's most often caused where certain patients may be more responsive to certain therapies and others. And I think by using this genome editing technology and IPS cells to look at, different risk factors in the same background, you can start to try to tease out, what are the similarities, what are the differences. As an example of this, some of the risk factors that have been identified recently that are the strongest, affect immune function. It's not actually neurons themselves, it's actually affecting the microglia. So, there may be people that are either suppressing or activating the immune system may be more important than other people have Alzheimer's. The second question, I'm not going to go into the details but we talked about how A beta and this model seems to be upstream of tauand drive Tau. Two decades later, they still do not know how this works. I find that amazing. And to me, that shows that we have some missing gap in knowledge from my perspective because we don't really mechanistically understand this link at least in a way that's universally accepted. The third one which I'll talk a little bit is how does AD pathology spread through the brain and what do I mean by that. This is in collaboration with Dr. Karen Duff. Also of Columbia. So, there's this kind of new newish theory that's come out the last few years that in a lot of neurodegenerative diseases not just Alzheimer's, you have these misfolded proteins that are aggregating and then the aggregation can spread through the brain and get proteins are not aggregated before to aggregate and also become toxic. So, the idea is you have some events that lead to this misfolded protein. This leads to a different structure that starts to aggregate. These aggregations can fragment and then, through some mechanism that isn't understood, there's multiple hypotheses but none are accepted at this point, that it can then spread to the next cell and essentially, start the process again in a healthy cell and spread through the brain. So, if you look at mouse models you can activate tauin one part of the mouse in with neurons and syntactically connected to, it will travel and spread through the brain and that's true also with Alzheimer's pathology. If you look at brains, it starts in the Entorhinal Cortex and then the Hippocampus, and then it spreads, to other parts of the brain. So, there is this idea of of this pathological spreading. So, we want to be able to try to study this in human cells but to understand how it works and potentially also to develop therapeutics to try to block this aggregation or block this spreading. So, it's not exactly the same but one could kind of conceptional think of this as similar to Mad Cow disease or Creutzfeldt Jakob disease. The variant form is where you get it from an infected animal. The non-variant form is just where your own cell mutates and this is where you have something called the Prion protein that people may have heard of that misfold and then, gets normal versions of the Prion protein to also, misfold. So, in the case of brain, there's people that ate a cow form of the prior protein that then changed the confirmation of their own cells. And a couple of decades later, unfortunately their brain is kind of like Swiss cheese and you have all these holes in it. So, it's this idea that the Prion protein can spread. Now, the mechanism may or may not be the same for tauin Alzheimer's but conceptually that's at least somewhat similar. So, how do we model this in a dish. So, one way we did this is we put in what's called the Reporter. So, reporters are ways of visualizing things easily. So, in this case we took part of the Tau gene. We got this from another lab that had part of Tau that was fused to a yellow fluorescent protein. So, even though it's yellow, if you shine a light on it, it makes a green. Acceptation. So, basically putting this reporter. So, here is control stem cells. We don't see any green cells. We put in the reportor, you can see these green cells and then, when we add a seed, in this case, another cell line where we put in synthetic Tau, we can get these little tiny bright green dots that you can see, and that is the protein aggregating. So, you can visually look at this action in real-time to see these kind of aggregation events. So, this is in stem cells, but of course, you also want to look in neurons, which are the ones affected. So, in this case we differentiated this line into neurons there for 42 days. So, they were at least moderately, electrically active. And what we can see with these little white arrowheads is that we have aggregation in the reporter. And the difference is this time instead of taking synthetic tauin a cell line, we actually took, patient brain homogenate from a late-onset Alzheimer's patient brain who had strong taupathology and we could take that person's brain and use that to seed and essentially, cause tauaggregation in our reporter. Furthermore, though the others possibly could be some other explanations and if you can see with the white arrowheads these little red dots. So, this is immuno sitting with something called MC1. So, this is a kind of late confirmational change antibody which will detect very changed in aggregated tauand we seem to be getting these in the process of our cells. So, consistent at least with us, seeding the endogenous tauthat's there, not just this reporter that we put in. So, I think this is a very useful system that we are going to be able to use to try to study how this process works better and then ultimately, hopefully, to try to stop it. So, what are my overall perspectives on this. So, I think personalised Pluripotent Stem Cells, whether generated by IPS technology or CRISPR Cas technology, is going to really revolutionize the study of disease. And I think, it's already starting to do so and provide much better screening platforms for drug development. Typically, drug screens in the past were mostly in animal cells. They weren't on human cells. They were immortalized cancer lines that were different genetically by definition. So, now we are taking cells that are not cancer cells, that actually are the cells that are affecting disease, albeit maybe an immature version of that cell, and we are essentially people are starting to do drug screens on what I think is a much better cell type. Also, in the long run, this is going to lead to personalized, autologous stem cell replacement where we make stem cells from people and use those cells to replace their own tissues. So, with that, I want to thank the TAUB family for supporting my lab, the members of my lab, our collaborators, and you for your attention.