University Place  Anita Bhattacharyya Using Human Stem Cells to Study Down Syndrome  April 27, 2013 UP869   cc >> Work using human stem cells to understand Down Syndrome brain development. Today, I'm going to give you a little bit of background before I jump into our work. I'll tell you a little bit about what we feel we know about Down Syndrome brain development at the cellular level. So this is very different from the talk you heard this morning talking about whole brain and different areas of the brain. Now we're getting microscopic and we're looking at individual cells. I'm also going to give you a little bit of background about human stem cells and the kinds of stem cells that we are using, and then I'll tell you about our recent work on using Down Syndrome human stem cells and what we've discovered so far. So, first of all, as you are aware, there are many differences in the Down Syndrome brain as compared to unaffected individuals. And there are differences, I don't expect you to be able to see this very well, but it just shows a schematic of a brain and shows that in different areas of the Down Syndrome brain there are different deficits, and these vary based on which part of the brain, and some of the regions are normal so there are very specific, very localized differences in Down Syndrome brain. And most of what we know about what is different about Down Syndrome brain is based on looking at adult brain, and, taken together, I've kind of distilled down what I think are the really three main neurological deficits that are found in Down Syndrome brain. And they are that there are fewer neurons or brain cells in certain parts of the brain. 

There are faulty synapses. This is how nerve cells communicate with each other. And, as we've already heard, Alzheimer's disease, our neural degeneration is characteristic of Down Syndrome. But it's been hard for us, oh, let me just show you some examples of this. So, these things are quite hard to study, how, at a cellular level, the brain of Down Syndrome individuals might be different. So I'll show you a couple studies, and this is what most of the evidence is based on. So brain development happens prenatally, the formation of the brain happens prenatally, and so using postmortem samples, some people have counted the number of neurons or brain cells in the brain and see that during gestation, or prenatally, the number of neurons really increases like you would expect as the brain gets bigger and forms. But in the small number of samples from postmortem Down Syndrome brain find that there are actually quite a bit fewer numbers of neurons in the brain, just again illustrating that there are fewer neurons in Down Syndrome brain. There aren't none and there are definitely more than half. So we have to remember that much of what we see in Down Syndrome nervous system is actually quite normal. 

Second, there's miscommunication between nerve cells because those synapses, those areas of communication or those spots of communication are not formed properly. So this is one axon of a neuron, and these little spines that you see on here are the actual synaptic contacts. That's where two neurons talk to each other via the synapses. And normally on a little part of an axon you have lots of these little spines which indicate lots of synapses. In Down Syndrome neurons you actually see that there should be fewer of these spines, and they look less developed. They're a little smaller and shorter, and this is a lot of the evidence for why we think that there is faulty synaptic communication in neurons in Down Syndrome. But, as I said, this is really hard to study. When does this start? What causes it? Is there anything we can do about it? Because the formation of the brain happens prenatally. And so by the time a baby is born, the formation of the brain, all the cells are where they need to be, and now the main job of the brain is to make the right connections so that it can function. We do know that the way the brain is formed is in a very specific pattern. 

So, from about 10 to 25 weeks gestation, all of the neurons that are in our brain are formed, are born and moved to where they need to be. And different kinds of neurons generated at different times along this timeline. And then after all the neurons are formed, then all of the support cells, like astrocytes and myelinating cells, are formed. So we know that this happens so we know where we would like to look when ask questions about neural development. But we can't look easily, again because this happens prenatally. So what I'm going to try and tell you today is how we can use human stem cells as a window to study prenatal development, and, more specifically, how the brain cells that carry the extra chromosome 21, how they develop differently from those that don't. So, first of all, I need to give you a little primer on what are stem cells. So stem cells, it's actually quite simple, they have two characteristics that make them unique. The first is that they can differentiate into any cell type of the body which makes sense because that's what their job was in the first place is to populate our body. So one stem cell can give rise to, say, a neuron, liver cell, and a skin cell. 

So that's the first characteristic. They are pluripotent. The second characteristic that makes stem cells unique is their ability to more of themselves, or self-renewal. And that, again, allows a single stem cell to populate an entire organism, but it also allows researchers to harness that capability and put these stem cells in a culture dish and grow them indefinitely. Once we learned how to keep these in a culture dish, then we could give them signals to keep themselves renewing again, generating millions and millions of stem cells that we can use. And then, when we want, we can then release those growth factor signals and differentiate them into different cell types. So there are three main types of human stem cells, which I'm not going to spend a lot of time on today. There are the human embryonic stem cells which you have undoubtedly heard about. These are cells that are derived from human embryos at about five to seven days after fertilization. There are also tissue specific or adult stem cells that we get from a developing or adult organ. 

These are not always considered true stem cells because they don't self-renew very well in culture. You run out of them. And also they only usually differentiate into some cell types. So a good example of this are bone marrow stem cells that we use for bone marrow transplantations. They are really good at making blood cells, and that's why they're used for a lot of blood disorders. They're not very good at making other kinds of cells in the body. And also, they are not very good at self-renewal. We can't grow them in a culture dish for long periods of time, and that's why we have to go back to donors every time we want to do a stem cell transplant. So, the last kind of stem cell is called a reprogrammed or induced pluripotent stem cell, and these are the ones we're using, and I'll tell you what they are. So, as I said, embryonic stem cells are derived from human embryos. 

They can differentiate into all cell types of the body, and in the last 15 years since Jamie Thomson first described how to isolate human embryonic stem cells, his lab and many others around the world have spent a lot of time trying to figure out what is it that makes the embryonic stem cell unique. And what makes it different from all of these other cells that it can differentiate into, and, also, what makes it different from a cancer cell which can self-renew as a stem cell does, but stem cells don't turn into tumors. So, based on all that research in the last few years, many scientists, including Jamie Thomson's lab and Shinya Yamanaka's lab in Japan discovered that there's actually a pretty simple recipe for taking a differentiated cell, like a skin cell, and turning it back into a cell that looks like a stem cell. And we do this by forcing the expression of specific stem cell genes in these cells. And so these cells now have been reprogrammed into a human stem cell, and we call these reprogrammed stem cells or induced pluripotent stem cells. So we've induced their pluripotency. And so far as we can tell in the five years since this has been reported, they behave very similarly to human embryonic stem cells. There are still some ways in which they're different, but for the purposes of this talk, so far it hasn't affected our research. So, again, we can now harness this technology by taking skin cells from individuals. We have done this via skin biopsies to get the skin cells. 

We take those skin cells and we reprogram them into these IPS cells, and then these IPS pluripotent stem cells we can differentiate into all cell types of the body. So while I'm going to talk to you about how we can use them to study brain development, they can be used to study any kind of cell type in the body because they are pluripotent. Reprogrammed stem cells have some obvious and not so obvious advantages over human embryonic stem cells. The main is that they avoid the ethical debate over using embryos because they're actually made from adult skin cells or from skin cells from an individual or blood cells, as I'll tell you about. The initial hype about induced pluripotent stem cells, IPS cells, was that you could take a person's skin cells, turn them into IPS cells, and if that person had a specific disease, you could fix the disease in those cells and transplant them back into the person. And so a patient's own cells could be transplanted back into them in cell therapies, and this would potentially avoid the rejection issues that come with cell therapies. This has not been realized, and actually there isn't, this is more a possibility but less has been done on it at the moment. But the most important thing is that IPS cells can made from people with specific diseases and disorders because you need just to get, you can get skin cells or blood cells from people who've been diagnosed with a disorder. And this has been especially useful for neurodegenerative disorders where the age of onset and diagnosis is often not until late in life. So we have made reprogrammed IPS cells from individuals with Down Syndrome using skin biopsies, and these pictures are just to show you that our Down Syndrome IPS cells, designated here with DS, look like human embryonic stem cells, and our unaffected IPS cells, they express the same markers, shown here in fluorescence, and, most importantly, when we reprogram these Down Syndrome cells, they retain the extra chromosome 21. 

We didn't mess that up in doing this reprogramming process. So now what we want to do is do what all other researches are doing with their IPS cells from their disease or disorder of choice. So, we can take skin cells again, or we have recently been able to do this same process from blood cells, and I think, as Marsha pointed out, we are recruiting for individuals with Down Syndrome to donate blood samples for the study. And I hope by the end of the talk I've convinced you that this is a worthwhile thing to do. So we can take skin cells or blood cells and turn them into IPS cells. And then we can grow these in culture, and what we're interested in doing is turning them into neurons and figuring out what's different about these neurons, and hopefully figuring out genetically what is causing that. We know it's due to the extra chromosome, but we need to know more specifically, is it one gene that's doing this, is it something we can block. And, also, these cells are often shared with other researchers that have other questions they're interested in via stem cell banks. So the first thing we did with these cells is we turned them into neurons, following protocols that have been well-established here at the Waisman Center by Su-Chun Zhang who really pioneered turning human embryonic stem cells into neurons and has now been able to do that with IPS cells. And the first thing we found out, to our joy, is that we could make very beautiful neurons from our Down Syndrome IPS cells as well as our controls, and they looked very similar. 

Here we've stained them in green, and the nuclei of the cells are stained in blue. So it's the green neurons that you're looking at. And when we counted them, the Down Syndrome IPS cells made the same number of neurons as the control IPS cells, and so this told us, at least in these kinds of neurons that we're studying, there isn't a difference. We're not getting fewer of them, at least at this point. Then we wanted to look at whether these Down Syndrome neurons had functional properties that were different And so what we wanted to do is take these neurons in the dish and ask whether their a synaptic activity and their electrical activity, that is how they communicate with each other, if it's different or similar. And to do this, we take these cells in the culture dish, they're actually not green when we look at them, and we stimulate them with an electrode, and then we record from them using a specialized pipette that forms a seal like a little suction on the outside of these neurons. And this shows you, I hope you can see this, here is one of the neurons in culture and here's a pipette coming in, and we have to go in and just touch it so that we can measure what's going on in that cell. And when we do that, we can see electrical activity, shown by these traces, in the neurons. So we did that for our Down Syndrome and control neurons, and we found that our neurons, first of all we found out that most of our neurons did have electrical or synaptic activity. And the representative trace from the control neurons is here, and the representative trace from the Down Syndrome neurons is here. 

And I hope you can see that there is a quite subtle difference in the Down Syndrome neurons, that they have fewer of these spikes. They have less activity, and, actually, when we quantified this, we found that fewer of the neurons had synaptic activity and, of those that did, they had a lower frequency. And so what this means is if you have a whole bunch of Down Syndrome neurons, fewer of them are communicating properly, and of those that are communicating, they might be doing it in a quieter way. And so if you're thinking about how the brain forms all these connections, if you have these Trisomy 21 neurons who might not be doing it quite as well, it can alter the way that network and that communication is established, even this early in development. And I'll tell you that these are neurons that have been in culture for about six weeks. So we're looking at early events, we think we're looking at very early events in neural development. We then looked more carefully at these neurons and found that that decrease in synaptic activity was mirrored by a decrease in the number of synaptic contacts on these neurons. And I know this might be a little bit hard to see, but here is a representative image of a control axon and here is a Down Syndrome axon. And this is similar to what I showed you before with these spines, but at this resolution we can't see spines but we can label the synapses with marker that has shown here in green. And then we get a bunch of undergraduates to count these little dots on these lines. 

[LAUGHTER] And we come up with a number that shows us that the Down Syndrome neurons have fewer of these synaptic puncta, or synapses on the neurons. And at this point, we've only looked at one time point. We don't know if this is a deficit that remains throughout development or whether this is just a delay and those Down Syndrome neurons will catch up if we give them enough time. But even if that is true, again, this is a crucial window of time where they're making connections with other cells. So, while a delay will definitely increase their function later, they may have missed, these neurons may have missed crucial opportunities to interact with other neurons. We also don't know if other neurons, we were looking at one kind of neuron, have these same synaptic defects. So the other thing that we found in these neurons is they have increased oxidative stress, and you hear a lot about oxidative stress just in the general population but also in Down Syndrome. This is something that's not necessarily new in Down Syndrome that Down Syndrome cells have increased oxidative stress, but we have not yet seen this before in human Down Syndrome neurons. And so what I'm showing you here is we use a specific dye, a red dye that shows when there is oxidative stress in a cell. And when a cell is feeling oxidative stress, which these Down Syndrome neurons are, that means that they're responding to things in their environment in a way that they're having trouble adapting to it. 

And so they have a lot of reactive oxygen within them. And what we found that not only do they have increased oxidative stress, but their mitochondria, the powerhouse of the cell, are not functioning well. Increased green dye in this particular case means that the mitochondria in the cell are not as healthy, they're not as happy. And so what we think is that these Down Syndrome neurons that we have in culture are really stressed to the same environmental conditions that don't seem to stress our control cells as much, and this may make them more vulnerable to degenerating. We don't see a lot of these neurons dying in our cultures, but we have an artificial system. We may be giving them a lot of good things so that they aren't dying, but they're definitely more vulnerable.  So, I want to, that's all of the data I have for you. I want to kind of come back to what I first told you about the three things that we focus on as being the neuropathology in Down Syndrome at a cellular level. I said that there are many areas of the Down Syndrome brain where there are fewer neurons, and I've shown you that we don't see, at least in our initial cultures, that these early born neurons, the neurons that we differentiate first from our Down Syndrome IPS cells, make normal numbers of neurons. So this is an area where the extra chromosome 21 is not causing them to make fewer neurons. 

We need to look at other neuronal subtypes to see whether those neurons that are born later, or even astrocytes or myelinating cells, may have some deficits. I told you and showed you that Down Syndrome neurons have faulty synapses, and I've shown you now that we are able to model this in our culture system where we see fewer synapses and less electrical activity in our human Down Syndrome neurons. And this is really critical because this is an area where it's a very dynamic process that's going on, electrical activity and synaptic activity. It's not something that you can look at in a postmortem brain slice. This is something you really want to manipulate in the culture dish. And so, by having the ability to model this in the culture dish, we can pinpoint better why do they have less synaptic activity. Is this a delay or a maintained deficit? And so, again, it enables us to really look at this dynamic process. And finally, I would propose that having the oxidative stress vulnerability that we see in our Down Syndrome neurons may contribute to degeneration and the synapse loss that we see in Alzheimer's disease. That's something that's very hard to study in a culture dish. If Alzheimer's disease is a disease of aging, do we have to keep our cells in culture for 30 years in order to see this? We don't know. 

Or do we just stress them in a way that would mimic the aging process? So we don't know. And we're actually working with Brad Christian's group to make IPS cells from individuals with Down Syndrome who are older and may be exhibiting some aspects of Alzheimer's disease. So, how can the information that I've shown you today really help us and help people with Down Syndrome? So, first of all, I would say human stem cells are not cell therapy for Down Syndrome. Nothing I've shown you and I have not seen any data that suggests that transplanting new neurons or more neurons or more cells into the brains of individuals with Down Syndrome would really have any effect. The bottom line is there are so many things that we don't know understand about what might be going wrong that we don't yet know how to provide therapy. So, I think that by using these cells as a way to learn more about the neurons as basic research, we can pinpoint the specific deficits that occur and we can find out when, and then we can more intelligently design therapeutics. The other thing that I think these cells are really good for is actually testing therapeutics that are out there or that will be developed because most drugs that are used now that are developed by pharmaceutical companies are tested in animal models and then they go straight to human clinical trials or early clinical trials. And the first kinds of clinical trials are usually safety because that's a big jump going from a rat to a human, and there are many things that are different, hopefully, and so there are often drugs that won't work. So, having these human neurons, as well as many other cell types that are derived from human stem cells, it provides us a way in a minimum to find out if those drugs that work fine in animals are toxic to human neurons, for example. And, again, provides a bridge between the animal models and the clinical trials so as, again, to better design some of the therapeutics and better use the drugs that are out there. 

So, I want to end with that and by thanking the people who did all the work. I am indebted to Su-Chung Zhang for developing most of the methods that I've shown you here and for his continued mentorship. And this work was mostly carried out by a postdoc and technicians and my army of undergraduates to look at puncta and cells and count the colors. And then we have a core facility here at the Waisman Center where we generate induced pluripotent stem cells, or IPS cells, from skin and blood samples. So we do this all in-house. So the way that it has worked for those of you who've done it, which includes Sterling, is that you come into the clinics, you get a skin biopsy, now you can get a blood draw, and those cells are taken upstairs to our stem cell labs, we reprogram them into these IPS cells, and then we can begin work on them. I want to acknowledge also my funders, and I am also very appreciative of the Waisman Center for the support that they've given me. And the stem cell research program here at the Waisman Center is part of the larger university Stem Cell and Regenerative Medicine program. And, as Marsha pointed out, most of the researchers who work on stem cells in the brain are located here. And my funding is from a very generous donor, the Phipps family who have a 50-year-old son with Down Syndrome, and also NIH and a French Down Syndrome foundation. 

So, thank you and I will take any questions. [APPLAUSE]