Biological Foundations of Mental Health PDF

Module: Biological Foundations of Mental Health Week 2 Building blocks of the brain Topic 2 From embryonic NPCs to AHN - part 1 of 4 Dr Brenda Williams Senior Lecturer in Neuroscience (Education) Lecture transcript Slide 2 In this short section, entitled From Embryonic Neural Progenitor Cells To Adu...

Module: Biological Foundations of Mental Health Week 2 Building blocks of the brain Topic 2 From embryonic NPCs to AHN - part 1 of 4 Dr Brenda Williams Senior Lecturer in Neuroscience (Education) Lecture transcript Slide 2 In this short section, entitled From Embryonic Neural Progenitor Cells To Adult Hippocampal Neurogenesis, I want to link what you heard from Professor Sarah Guthrie about the generation of neurons, or neurogenesis, during development, to what you will hear from Dr. Sandrine Thuret about the generation of new neurons in the adult brain. As you will remember from Sarah’s lecture, during development neurons are generated by radial glial cells. Here we can see a picture of radial glial cells in the developing mouse cortex. In this picture, these cells appear green because they have been labelled with a green fluorescent protein so that they can be observed under a fluorescence microscope. These cells get their name because of their radial morphology. Their processes span the entire thickness of the developing cortex, from the ventricle, labelled "V", at the bottom of the picture to the peel surface, labelled "P", at the top of the picture. And also because they share certain characteristics with a particular type of glial cell in the brain called an astrocyte. During development, these radial glial cells are generated from new epithelial cells. These cells are the cells that form the neural tube and are the characteristics of embryonic neural stem cells, as I will explain shortly. The picture shows a section through a neural tube with the new epithelial cells stained using an antibody that recognises a protein called SOCS-2. This protein is expressed by new epithelial cells. These cells appear pink in colour because the antibody has been tagged with a fluorescent probe so that these cells can be observed under a microscope. Later in development, these radial glial cells go on to generate adult neural stem cells as required to generate specific types of neurons in the adult brain. The stem cells reside in two specific locations in the adult brain, the subventricular zones of the lateral ventricles and the subgranular zone of the dentate gyrus, which is part of the hippocampal formation. You will shortly hear more about adult hippocampal neurogenesis from Dr Sandrine Thuret, but what I want to do now is to give you an overview of how we get from the new epithelial cells of the neural tube to the adult neural stem cells via radial glial cells. Week 2 © King’s College London 2019 1. Before going any further, I would like to remind you that further information on any of the concepts that I cover here can be found in the references provided to you for this sub-topic. Slide 3 As I mentioned earlier, the new epithelial cells that form the neural tube are the founder cells of the central nervous system, and as such, can be thought of as embryonic neural stem cells. This means that they have the capability to generate all the different cell types in the developing central nervous system. That is, they have the ability to generate all the different types of neurons, and also, two types of glial cells, astrocytes and oligodendrocytes. All the pictures on this slide show you what these different cell types look like when isolated and grown in the laboratory. Again, they appear coloured because they are labelled with specific markers that have been tagged with a fluorescent probe, allowing these cells to be observed under a microscope. Slide 4 Like all other stem cells, embryonic neural stem cells are non-specialised cells that have two specific characteristics. They can self-renew and differentiate. They differentiate into appropriate specialised cell types, which for neural stem cells are neurons, astrocytes, and oligodendrocytes. Let’s look at each of these characteristics in a little more detail. Self-renewal is the ability of a cell to divide and generate two cells that are identical to the parent cell. Self-renewal is needed to make sure the cells don’t run out. In other words, that sufficient numbers of embryonic stem cells are present to enable the generation of all the different brain cells that we need. Differentiation is the ability to divide and generate more specialised cell types. This process is important for making all the different kinds of cells that are required to generate a proper functioning brain. Differentiation can occur in a number of ways. To explain this, I will consider the different ways that neurons may be generated. An embryonic neural stem cell may divide and generate another embryonic neural stem cell and a neuron. Or an embryonic neural stem cell may divide, generating a progenitor cell, like a radial glial cell, and a neuron. This radial glial cell also has ability to self-renew, but it does this mainly by dividing to generate one cell that is like itself and a neuron, but the radial glial cell might also divide to generate a dedicated progenitor cell. That is, a progenitor cell that has the ability to only generate a single cell type. For instance, a neuron. And while doing this, it also generates a neuron. This process of differentiation, where a parent cell makes two different progeny, is called asymmetric differentiation. However, radial glial cells cannot make embryonic neural stem cells, and dedicated progenitor cells cannot make radial glial cells or embryonic neural stem cells. And as I’m sure you know, neurons are terminally differentiated, so do not divide at all. Week 2 © King’s College London 2019 2. Slide 5 So if we think about specialisation, the embryonic neural stem cell is the least specialised cell. Then we have the radial glial cell, then the dedicated progenitor cell, and then the neuron. Slide 6 Let me now put these ideas together and link these to the generation of adult neural stem cells. Initially during development, embryonic neural stem cells self-renew to expand the progenitor pool. They will then begin to generate neurons, because during development, neurons are generated before glial cells. As well as generate neurons, embryonic neural stem cells will also generate radial glial cells. These cells, as I told you, also have the ability to self-renew and generate neurons either directly or via a dedicated progenitor cell. That is, a cell that will only generate neurons in this case. Just to complicate matters further, embryonic stem cells can also generate neurons via dedicated progenitor cells, too. Why are there so many different ways to generate neurons? Well, we consider that this is because there are many different types of neurons that need to be made over a very specific time period during development, and this is especially true when we consider the complexity of the human brain. Later in development, radial glial cells begin to generate oligodendrocytes and astrocytes, again via dedicated progenitor cells. Radial glial cells also generate another type of cell, the adult neural stem cell. As their name implies, these cells retain the capacity to generate new neurons throughout our lifetime. You will now explore the characteristics and function of adult neural stem cells with Dr Sandrine Thuret. Week 2 © King’s College London 2019 3. Module: Biological Foundations of Mental Health Week 2 Building blocks of the brain Topic 2 From embryonic NPCs to AHN - part 2 of 4 Dr Sandrine Thuret Department of Basic and Clinical Neuroscience Lecture transcript Slide 2 So you have just learned about the concept of neural stem cells and the production of neurons derived from these neural stem cells during development. Now we are going to go through the concept of adult neurogenesis, then explore the location/environment - also called the ‘niche’ - where adult neurogenesis is occurring. I will also discuss the molecular control of adult hippocampal neurogenesis, the functionality of adult hippocampal neurogenesis, and finally how adult hippocampal neurogenesis can be modulated. Slide 3 So the concept of adult neurogenesis, or the birth of a new neuron in the adult brain, is fairly new. And since the early 1900s, it was generally believed that no new neurons can be generated in the adult central nervous system. And, as then stated Cajal, ‘Once development has ended... everything may die, nothing may be regenerated’. Then, 50 years ago, Altman and collaborator suggested, with autoradiographic and histological evidence, that some new neurons were indeed born in the adult hippocampus of rats. But it is not until the early ‘90s that work started again, with the development of new technology, to definitely prove that neurogenesis was occurring in restricted regions of the adult brain. Slide 4 So two adult neurogenic niches have consistently been found in the rodent, during normal physiological conditions - the subventricular zones of the lateral ventricle, where the neural stem cells give rise to newborn neurons that will migrate to the olfactory bulb, and the second niche is the subgranular zone of the dentate gyrus in the hippocampus. Importantly, the generation of new neurons throughout adulthood has not only been demonstrated in the hippocampus of rodents but also in the hippocampus of humans. Slide 5 Indeed, elegant work from the group of Jonas Frisen, from the Karolinska Institute, assessed the generation of hippocampal cells in postmortem human brains by measuring the concentration of nuclear-bomb-test-derived C14 in genomic DNA. In this figure, extracted from the original article, we can see C14 concentration in the hippocampal neurogenomic DNA correspond to a time after Transcripts by 3Playmedia Week 2 © King’s College London 2019 1. the date of birth of the individual, demonstrating neurogenesis throughout life. Slide 6 So when we zoom in to the dentate gyrus of the hippocampus, and then, in the granular cell layer, we have our neural-stem-cell niche, where they will proliferate, differentiate and mature into neurons through the granular cell layer, where they will mature and receive input from the entorhinal cortex and extend projection into the CA3. And it will take up to four to six weeks to go from neural stem cells to mature neurons in rodent, as we see here, on the figure on the right. Slide 7 So how relevant is the amount of newborn neuron generating during adulthood? In the adult hippocampus, in human, it is estimated that we produce around 700 new neurons in each hippocampus per day. It does not seem a lot, among the billions of neurons we have in the brain, but by the time we turn 50 we will have replaced the entire granular-cell population we were born with, with adult-born neurons. When investigated adult rodent and a very particular neurogenic niche, it has been found that 70% of the bulbar neurons are replaced then during a six-week period. Slide 8 So, now, what makes both two niches so special? Why neural stem cells from only in those privileged areas of the adult brain can generate neurons? What environment makes neurogenesis possible? Slide 9 So there has been some classic transplantation studies providing direct evidence for the regulation of fate determination by extrinsic signals that are derived from the neurogenic environment. Such as, if you extract neural stem cells from non-neurogenic regions, like the spinal cord, then grow them in a dish, expand them, then take these cells you have grown in the dish, and then you transplant them back into a non-neurogenic region - so, back into the spinal cord - you still do not get any neurons. However, if you take them, grow them in the dish, and then transplant them in a neurogenic region like the dentate gyrus or the subventricular zone, then these neural stem cells issued from a non-neurogenic region, transplanted in a neurogenic region, give rise to neurons. Slide 10 Conversely, if you take neural stem cells from a neurogenic region - let’s say, from the dentate gyrus - then transplant them in a non-neurogenic region, like the spinal cord, you do not get neurons. Of course, you transfer them back in a neurogenic region, like the dentate gyrus or the SVZ-- the subventricular zone - then you get a neuron. So, demonstrating direct evidence for the regulation of neuronal fate, determination of stem cells by extrinsic signals that are derived from the neurogenic microenvironment by the niche. Slide 11 So, what constitutes a neurogenic niche? We know that endothelial cells and proximity to blood vessels do play a critical role but that also astrocytes within the niche are very important. Slide 12 And I would like to highlight here, the first article by Song and collaborator, from the Gage lab, providing evidence that astrocytes are key players in the neurogenic niche, to instruct neural stem cells to adopt a neuronal fate. Transcripts by 3Playmedia Week 2 © King’s College London 2019 2. So what they did is co-culture experiments. So they extracted neural stem cells from the adult hippocampus - so, here, labelled in green-- and then co-cultured them with astrocytes, either extracted from the adult hippocampus or with astrocytes extracted from a non-neurogenic region, like the spinal cord. So, leaving those neural stem cells to differentiate, together with these astrocytes in cultures, so they did produce more neurons - so, then expressing this neuronal marker mapped to in red - when co-cultured with hippocampal astrocytes. So it is nicely quantified on the graph, where the neural stem cells produced the most neurons when co-cultured with neonatal hippocampal astrocytes - so, young hippocampal astrocytes. Then the next-best were adult hippocampal astrocytes, and then with spinal-cord-derived astrocytes leading to the lower number of neurons - comparable, actually, to control condition - without any astrocytes. Transcripts by 3Playmedia Week 2 © King’s College London 2019 3. Module: Biological Foundations of Mental Health Week 2 Building blocks of the brain Topic 2 From embryonic NPCs to AHN - part 3 of 4 Dr Sandrine Thuret Department of Basic and Clinical Neuroscience Lecture transcript Slide 2 So, we have learned about the niche. We have learned that astrocytes are important. But what are the actual molecular controller responsible for adult neurogenesis? Slide 3 So this is another view of what we knew about the molecular control of adult neurogenesis ten years ago. This figure is quite sparse. At the time, we understood very well how adult neural stem cells proliferate - with, for example, epidermal and fibroblast growth factors, EGF and FGF2, which are the primary mitogens used to propagate neural stem cells in vitro and are believed to be very important for the control of in vivo proliferation of neural stem cells or progenitor cells. Next, the molecular mechanism underlying fate specification of adult neural stem cells over a decision to actually become neuron at this time had just began to be revealed. So we knew, back in 2004, that adult neural stem cells express member of the bone morphogenic protein, BMP, family that instruct them to adopt a glial cell fate. However, in the neurogenics niche, the BMP inhibitor Noggin is secreted by the ependymal cells and presumably serves to block the gliogenic effect of BMPs, so driving the fate of the neural stem cells towards a neuronal fate in the niche. When we think about factor control in later step, in neurogenesis, such as functional maturation, synapse formation and integration into the neuronal circuit and the survival, were then, at the time, unknown. Slide 4 We now know a lot more on the molecular mechanism controlling all the steps of adult neurogenesis. And I invite you to have a look at this updated review and find your favourite molecule involved in proliferation, differentiation, migration, all the way down to integration. And for this lecture, I want to give the example of Wnt signalling as one of the key molecular regulator of adult neurogenesis. I chose Wnt, as it is actually secreted by astrocytes, one of our niche key player. Week 2 © King’s College London 2016 1. Slide 5 So in this 2005 Nature article, the author showed for the first time that Wnt signalling regulates adult hippocampal neurogenesis. The first clue they got was via in situ hybridisation, identifying cells expressing Wnt in the subgranular zone, where neural stem cells reside within the hippocampal niche. They then extracted adult hippocampal stem cells and cultured them in vitro, with or without Wnt factors. They show here that Wnt-3 pushed them towards a neuronal fate, as indicated by the increased number of neuroblasts, or young neuron, labelled here with doublecortin, or DCX, in red. It is nicely quantified on the graph, with a fourfold increase of neurons produced when neural stem cells are cultured in the presence of Wnt. Slide 6 For their next experiment, they moved in vivo and injected the hippocampus with a controlled antivirus only expressing a green fluorescent marker, or they injected the hippocampus with a dominant negative Wnt antivirus blocking Wnt signalling. They show here that the number of newborn neurons has decreased of eightfold when Wnt signalling was blocked, their data demonstrating that Wnt signalling was an important regulator of adult hippocampal neurogenesis. Slide 7 So we have gone through the importance of the niche. We have looked at some of the molecular control. So now, what is the functionality of adult neurogenesis? What are these newborn neurons for? Slide 8 So we know that adult hippocampal neurogenesis is important for learning and memory. The level of neurogenesis in the dentate gyrus is positively correlated with hippocampal-dependent learning tasks. And there is plenty of paper out there showing that type of evidence. And in many of the studies, if we actually block neurogenesis, then we block hippocampaldependent learning abilities. And the dotted line I placed there is to illustrate that actually hippocampal-dependent learning can also modulate neurogenesis - so, showing a bi-directional link between learning and neurogenesis. So multiple mechanisms for the relationship between increased neurogenesis and improved cognition have been suggested, including computational theories to demonstrate that new neurons increase memory capacity, reducing difference between memories - what we call ‘pattern separation’ - or add information about time to memories. Of these, post-natal new hippocampal neurons could be also involved in forgetting during infancy. So this is a new field, and we are still trying to understand the implications of this new neuron in memory formation. So research is still ongoing, to explore their exact role. Slide 9 Adult hippocampal neurogenesis is also implicated in mood regulation and depression. So, adult hippocampal neurogenesis is reduced in many animal models of depression, and many treatments for depression actually promote adult hippocampal neurogenesis. So, although more evidence suggests that neurogenesis alone cannot mediate the effect of anti-depressant, it is a key player. Such as, if you give anti-depressant to an animal model of depression, you will alleviate the symptom of depression. But if you block neurogenesis in the same animal model of depression, then you will prevent the efficacy of the anti-depressant - so, showing Transcripts by 3Playmedia Week 2 © King’s College London 2016 2. a link between neurogenesis and depressive behaviour. And research is still ongoing to understand more precisely the role of this new neuron in mood and depression. Week 2 © King’s College London 2016 3. Module: Biological Foundations of Mental Health Week 2 Building blocks of the brain Topic 2 From embryonic NPCs to AHN - part 4 of 4 Dr Sandrine Thuret Department of Basic and Clinical Neuroscience Lecture transcript Slide 2 So can we modulate neurogenesis? And if so, how? Slide 3 So here is a little quiz for you. So think if the behaviours I will give you can increase hippocampal neurogenesis in the adult or decrease adult hippocampal neurogenesis. You will have a few seconds to think in between each behaviour I’m going to present you. So what do we think about learning? Will it increase neurogenesis or decrease neurogenesis? Yes, learning will increase neurogenesis. How about stress? Stress will decrease the level of adult hippocampal neurogenesis, especially chronic stress. How about social interaction? Social interaction will be associated with a higher level of adult hippocampal neurogenesis. And conversely, in rodents, social isolation is going to lead to decreased neurogenesis in the hippocampus. How about chronic sleep deprivation? Yes, chronic sleep deprivation is going to decrease the level of neurogenesis in the hippocampus. What do we think about running and exercise? Running will increase the level of adult hippocampal neurogenesis, and I will highlight that in a few slides. And finally, what about getting older? As we get older, neurogenesis is still occurring. However, the rate of neurogenesis is going to decrease as we age. Slide 4 And this is beautifully illustrated by the work from Villeda, et al. So this is an example of how ageing influences adult hippocampal neurogenesis. And more precisely, how the ageing systemic milieu, or blood and serum, modulate adult hippocampal neurogenesis. So this is a 2011 Nature article by the lab of Wyss-Coray using a parabiosis approach, which means Week 2 © King’s College London 2019 1. fusing the circulatory system of an old and a young mice. So they have isochronic control - young and young in yellow. We have an old and old isochronic pair in grey. And then they have their heterochronic young, old pair in the middle. And they let them attach for three months and then look at their brain and look more precisely at the adult hippocampal neurogenesis. So what we see when we look closely, and they label the newborn neurons or neuroblasts with doublecortin, and we see that in the isochronic mice - young and young - we have a nice level of neurogenesis. Then when we look at our heterochronic young brain that was fused with an old brain, we see that there is a decrease of the level of neurogenesis. When we look at how old isochronic pair - both brains - we see that there’s a dramatic reduced level of neurogenesis. But then if we look at the heterochronic old brain that was fused with the young animal, we see that compared to the old animal it has an increased level of neurogenesis. And it is nicely quantified in the C section of the figure and D section of the figure, where we see that our young mice that was fused with old mice has a decreased level of neurogenesis. Whereas the old mice that was fused with a young animal actually has an increased level of neurogenesis. And they could recapitulate that experiment by simply injecting the plasma. So if we look on the right side of the figure, and they take a young mice as a control and then inject young mice with the young plasma, and then they take an old blood plasma that they inject into young mice. Then we look at the level of neurogenesis. And you see that the young mice injected with the young plasma have a normal level of doublecortin of neuroblast. But then if you look at the brain of the young mice injected with the old plasma, you see this dramatic decreased level of neurogenesis. And we could correlate that with, actually, their learning and memory abilities. Slide 5 So because of the functional role of adult hippocampal neurogenesis on learning and memory, and mood and depression, and the fact that we can modulate adult hippocampal neurogenesis, we think that neurogenesis in the adult hippocampus emerges as a target of choice to enhance learning and memory and mood, or prevent their decline. So we have already done a quiz and know how neurogenesis can actively be modulated, such as with running. Slide 6 So now, I want to highlight this article published in the Gage Lab - from the Salk Institute showing for the first time that neurogenesis can be modified by an intervention such as running. Slide 7 So here we see the hippocampus of a control mouse not having access to a running wheel. The cells labelled in black are neural stem cells proliferating that will lead next to neurogenesis. And compare this to the amount of black cells in mice who did run in D. We have nearly an increase of 30%, so showing that running is probably a very efficient intervention to increase the level of neurogenesis in the adult hippocampus. Slide 8 So let’s summarise what we have seen so far. So we know that learning, exercise is going to increase neurogenesis. We have not discussed particularly enriched environment. But be aware that by enriched environment, I mean putting toys, for example, in cages of rodent will increase the level neurogenesis, and in line then will increase their learning and memory abilities and improve their mood. Diet also can have a positive impact, but equally another type of diet can have a negative impact. We talked about ageing can decrease the level of neurogenesis. Chronic stress will decrease Week 2 © King’s College London 2019 2. neurogenesis. Chronic sleep deprivation will decrease the level of neurogenesis. Slide 9 So what we do can have an impact, but also what we eat can modulate the production of new neurons. Slide 10 So here you see all the diets that have been shown to modulate neurogenesis, and I’m just going to point out a few. So limiting calorie intake of 30% or doing intermittent fasting - so eating every other day - increased neurogenesis. Flavonoids contained in cocoa and fruits with dark skins like blueberry will increase neurogenesis. Omega-3 fatty acid contained in oily fish like salmon will increase the production of new neurons. Conversely, diets rich in saturated fat will decrease neurogenesis. Alcohol will also be detrimental to the production of new neurons. However, resveratrol contained in red wine has a positive effect. So now for a quirky one. There are entire groups of Japanese scientists fascinated about the role of food texture, and they have shown that soft food will decrease neurogenesis. So these data are derived from animal work, but actually the same diets that have been shown to impact memory and mood in human studies, and food modulates behaviour in the same direction as foodmodulated neurogenesis, such as decreasing calorie intake, intake of flavonoids, Omega-3 fatty acid, has increased the production of new neurons, will improve cognition and mood. Conversely, diets rich in saturated fat will decrease learning and memory abilities and exacerbate symptoms of depression. And some food seems to be linked to poor learning and memory abilities. Slide 11 Therefore, we have more and more evidence that suggests that neurogenesis mediates the effect of diet on mental health. Week 2 © King’s College London 2019 3. Module: Biological Foundations of Mental Health Week 2 Building blocks of the brain Topic 3 Exploring mental health using stem cells: What are iPSCs? - part 1 of 3 Professor Jack Price Department of Basic and Clinical Neuroscience Lecture transcript Slide 4 OK. So, as I said in my introduction, what we’re going to talk about today is stem cells and how they’re relevant to the study of diversity in brain and diversity that leads to neurodevelopmental disorders. And the stem cells I’m going to talk about are stem cells that we call iPSCs. So, iPSC stands for induced Pluripotent Stem Cells, and we’re going to describe what iPSCs are and how you make them. So the key word, really, in the title is pluripotent. So what does pluripotent mean, and why is it important? Slide 5 So pluripotentiality-- to understand pluripotentiality, we really have to go back right to the start of embryonic development in mammals, including humans. So to remind you of some sort of basic embryology, life starts with a fertilised egg-- sperm fertilising the oocyte. This fertilised egg grows, divides, forms a morula, and eventually it forms the structure we call a blastocyst. And a blastocyst is the first stage in development where you can see two distinct populations of cells. So there are these outer cells here, the trophoblast cells, shown in yellow in this diagram. But the cells we’re interested in are the blue ones. And these are the cells that we call the inner cell mass. And it’s these blue inner cell mass cells that we’re interested in, because these are the cells that are pluripotent. And what pluripotent means is that these cells have the ability-- the capacity-- to generate all the different cell types that make up both the foetal and then, later, the adult body. So, they can generate the immune system, the nervous system, the heart and circulatory system, muscles, and all the rest. So, that’s the definition of pluripotent. These are cells that can make everything that makes up the body. Now, the other point about this idea of pluripotency-- although it’s the most important concept you can imagine, really, in embryology, the property itself is incredibly ephemeral. So, these inner cell mass cells have this property of pluripotency, but they only have it for a few days, and then they lose it as these cells give rise to derivatives that are going to go on and make these different lineages of cells that make up the body. Transcripts by 3Playmedia Week 2 © King’s College London 2019 1. So, it’s a very ephemeral property. It’s there for a few days, then it disappears. And as far as we know, it never really reappears again during the entire life cycle of the organism. There’s some dispute about that-- there might be some populations of pluripotent cells crop up in other circumstances later. But, by and large, it looks as if these inner cell mass cells, for these few days, are the only cells that are ever pluripotent. Now, that makes it a very interesting property, needless to say, for an embryologist. But it also makes it a very difficult property to study, because if a property is only held by a handful of cells for a very short period of time in development, how on earth are you going to start to understand what it means in biological terms. And the real key to progress in this area has been the development of what we now call ES cells-Embryonic Stem cells. Embryonic stem cells were first derived from mouse, but have subsequently been derived from lots of different species, including, now, human. And the key here is-- what ES cells are is a population of stem cells derived directly from the inner cell mass. And the key was the generation of culture properties that assured that the ES cells retained this pluripotency. So, whereas for the inner cell mass cells, pluripotency is this ephemeral property that only exists for a few hours or days, in the ES cell lines derived from the inner cell mass cells, it’s a permanent property. ES cells are permanent cell lines that have this property of pluripotency forever. So, you can grow up as many of these cells as you like, and keep growing them, keep referring back to them. But these cells are pluripotent in exactly the same way as inner cell mass cells are, and that means, of course, just like the inner cell mass cells, they can give rise to all these different cell types that make up the body. Slide 6 So, what’s the biological basis for pluripotency? Well the answer is that until very, very recently, we had a very poor grasp of that. But the experiment that gave us the first clue as how to go about looking for the biological basis for pluripotency came from an experiment done by this gentleman, John Gurdon-- then at Oxford, now in Cambridge. And what he did was this experiment. He was working on frogs, and he took eggs from frogs-- so, an egg being the pluripotent cell of a frog, just equivalent, as it were, to the inner cell mass that we were just discussing. But what he did was he destroyed the nucleus of those egg cells. So, he used radiation, which would destroy the DNA. And sure enough, he was able to generate what he called enucleated eggs. So, these are pluripotent egg cells missing a nucleus, now. What he does then is he takes cells from fully differentiated tissue-- in this case, the skin. So, he takes skin fibroblasts-- also from frogs-- and he does the reverse experiment. So, what he does now is he takes the nucleus from those skin cells, and then the experiment is to combine this nucleus with this enucleated egg. So, what he’s got now is an egg with a nucleus transplanted from this fully differentiated skin cell. And the question, obviously, is, can this egg-- this egg with a transplanted nucleus-- go on and develop into a tadpole. In other words, is this cell-- this constructed cell-- is it pluripotent? And the answer was that it was. So, he was able to show in a convincing number of these circumstances where he transplanted the nucleus into the egg-- he was able to show that in fact that cell could go on and form tadpoles. So it was, indeed, pluripotent. Transcripts by 3Playmedia Week 2 © King’s College London 2019 2. Now, what do we conclude from that? There was a number of different conclusions to do with the fact that it shows that this nucleus, even though it’s from a fully differentiated cell, nonetheless still has all the genes-- all the information required to generate an entire organism. So, that was actually the primary reason he did the experiment. But we can actually take another conclusion from this. What we can conclude is the following. If we ask, where does pluripotency reside, it’s clear that it’s coming with the enucleated egg. In other words, the pluripotency is captured here, in the cytoplasm of this enucleated egg. And we can conclude that because this cell has been able to instruct this nucleus-- that was derived from the differentiated cell-- to act in a pluripotent manner. So, in other words, we can conclude that there must be factors in this enucleated egg that tell this nucleus-- once the two become combined-- that tell this nucleus that it’s got to start turning on the genes, behaving in a way appropriate for a pluripotent cell. So, we can conclude there are factors in the cytoplasm of pluripotent cells that dictate pluripotency. That’s a very important conclusion, and it allows us to beg the next question, which is, what are those factors? Slide 7 So, the Gurdon experiment told us there must be factors in the cytoplasm of that pluripotent cell that capture the essence of pluripotency. So the question is what are those factors. So, the real breakthrough in understanding those factors comes from the work of this gentleman, Shinya Yamanaka, in work that he published in 2006 and then again in 2007. And his approach to this problem was the following. He thought that the factors-- first off, they must be, themselves, gene products. In other words, they must be proteins. And he looked at the literature, looking at what had been published about pluripotent cells, and he came up with a list of 24 factors that seemed-- from the research of others and from himself-- that seemed to be associated with pluripotency. So, these are genes that seem to always be expressed, or always be around, when pluripotency was being studied. So, he figured that probably somewhere in this list of 24 factors must be the ones-- the really important ones-- that are dictating pluripotency in pluripotent cells. The problem, of course, is how is he going to show that the 24 factors do indeed include the important ones, and secondly, how is he going to find out which of those 24 really are important, and which, perhaps, are less important. So, this was the experiment that he set out to do. Slide 8 So, like all good biochemists, he realised that step number one-- he had to have an assay. He had to have a way of recognising if he’d produced pluripotency in cells that weren’t pluripotent. And just like John Gurdon, he chose to start with fibroblasts-- so, again, skin fibroblasts. And the challenge was, could he make those skin fibroblasts become pluripotent. And if he did, his assay would have to be, how would he recognise that had happened. And the key for him was to use this construct. So, what this is is a gene, Fbx15. And the actual nature of the gene doesn’t really concern as very much today. What’s important is, he recognised that pluripotent cells always seem to have this gene active. So, he figured that if he could switch on this gene in fibroblasts, then maybe he’d actually come up with a strategy that made them pluripotent. In other words, he was using this Fbx15 locus as a reporter, as we would term it, for pluripotency. Transcripts by 3Playmedia Week 2 © King’s College London 2019 3. So, he made this construct whereby if this gene became active, it would turn on this reporter construct that we call beta-geo. And as a consequence of that, if you stain the cells appropriately, they turn blue. So the experiment, now, is can he-- by putting those 24 factors into the cells, into fibroblasts-- can he turn on this beta-geo construct? Can he turn the cells blue? So, what he next did is actually a real tour de force-- so, a really impressive piece of molecular biology, because first off, he had to make retroviral constructs of all of those 24 genes. He had to have a way of getting all 24 genes into these fibroblasts. So he made 24 different retroviral constructs. He then had to engineer a situation where he could infect the cells with all 24 genes simultaneously. And what he found was the following-- that if he was able to introduce all 24 retroviruses into a population of fibroblasts, then some of them did indeed turn blue. And that’s shown over here. So, this is a plate of fibroblasts at very, very low power, so you can’t see individual cells. And these are what’s called the mock-infected. So, these are the control cells. But this is the plate of cells that were infected with all 24 retroviral vectors. And what you can immediately see-- and what Yamanaka immediately saw-- was that there are individual colonies that have started to emerge, and they’re stained-- they’re stained blue. So, in other words, by transducing in all 24 factors, he’d been able to show that some of those transfected fibroblasts turned on the Fbx15 locus, and therefore, putatively, the idea would be that maybe they had become pluripotent. So, just that step is an amazing step forward, because what he’s shown is that his first bet is correct. Somewhere in that 24 factors are the important ones that induce pluripotency in otherwise non-pluripotent fibroblasts. Slide 9 So, so far so good. But now he’s got another problem. How is he going to work out which of the 24 are really necessary, and which are perhaps not necessary? So, what he takes on now is an enormous experiment. So, what he’s going to do now is he’s going to repeat the experiment I’ve just shown you. He’s going to repeat the infection of these fibroblasts. But what he’s going to do-- instead of infecting with 24 factors, he’s going to infect with 23 factors. And he’s going to do it over and over, each time leaving a factor out. And the argument is that if it leaves out a factor that really isn’t necessary, then he should still see blue colonies, whereas if he leaves out a factor that’s important, now he won’t be able to generate the colonies. So, now what you’re looking at here is a whole series of experiments where he’s used the 24 factors minus one. So, each one of these experiments, he’s used 23 factors-- the 24 minus one. And what you can see, indicated by the green arrows, are circumstances where leaving out a factor made no difference. What you’re looking at here is the number of colonies he was able to observe-the number of blue colonies that emerged in these fibroblasts. And you can see leaving out factor 2, you still get lots of colonies. So factor 2 clearly isn’t very important. And similarly, these other factors, indicated with green arrows-- he could leave those out and the experiment still worked. So those weren’t necessary to induce pluripotency. But you can also see that there are some factors that, if he leaves them out, the experiment no longer works. So if he leaves those out, he doesn’t get any blue colonies. So, the conclusion is that those factors are probably necessary to induce pluripotency. Transcripts by 3Playmedia Week 2 © King’s College London 2019 4. Slide 10 As a consequence of that experiment, he takes-- he’s able to narrow down these 24 factors down to 10 factors. These are the 10 most promising-looking factors that emerged from this experiment. So, what does he do now? Well, he repeats the experiment that I’ve just shown you, but this time using 10 factors. So, first off he confirms here that by adding all 10 factors he does indeed induce pluripotency-- he gets colonies. So, what he does now is he leaves out one of the factors again. So, each of these experiments is one of the 10-- is the 10 factors with one factor missing. And again, what you can see is that in some cases he can leave the factors out, and it makes no difference. But there were four factors-- four factors that, if he left factor out, either there was no reprogramming to blue cells, or-- in the case of this one here-- there was some, but it was very much less efficient. So, a much reduced efficiency. So, now he’s down to these four factors. And these are the four factors we’ve now come to call the Yamanaka factors. So, Oct3/4, Klf4, Sox2, and c-Myc. And it seems like these are the four that really matter. And to confirm that, he does this experiment. So, now what he shows is that just those four factors still give him lots of colonies. So, if he uses just the four, he still gets conversion of the cells into blue cells-- so, putatively, pluripotent cells. And now, if he leaves out any one of those four, it works much less efficiently. And any two factors really don’t work at all. So, he concludes from that that these four factors are all necessary to generate pluripotent cells, and the four factors together are sufficient on their own. He doesn’t need any other of the 24 factors, and nothing else is required to generate pluripotent cells. Slide 11 So, this is a remarkable breakthrough, because he’s been able to identify four factors that seem to carry this property of pluripotency. But hold on-- all he’s shown so far is that those four factors can turn fibroblasts blue using that reporter construct. It’s still only a hypothesis that those blue cells-those cells that turn on the blue gene-- truly are pluripotent. To convince himself and other scientists that those cells really were pluripotent, he has to really show that they really can do the job of generating all the different cell types that make up the body. So he does that, first of all, by generating what we call embryoid bodies. So, if you take pluripotent cells-- for example, embryonic stem cells-- and you get them to grow in clusters, and treat them in an appropriate way, they’ll start to differentiate into little clusters of differentiated cells. And amongst these clusters, you will find all the different lineages that make up the body. In particular, you’ll find derivatives from what we call each of the three major germ layers-- that’s the endoderm, the ectoderm, and the mesoderm. And this is generally taken to be a good in vitro assay of pluripotency. Slide 12 So the question was, then, could he take these fibroblasts that have been transduced with the four factors, grow them as embryoid bodies, and show that each of the three germ layers were represented within the embryoid bodies. And what you can see here, in this figure, is the evidence Transcripts by 3Playmedia Week 2 © King’s College London 2019 5. that he can do exactly that. So, these are histological sections through embryoid bodies derived entirely from those transduced fibroblasts. And what you can see is that there are mesoderm derivatives, like muscles and smooth muscle, and cartilage, there was epithelial tissue, there was brain tissue, there was adipose tissue. In other words, these cells had managed to go on and make all of these different cell lineages. So, that was remarkable. It seemed to show that those cells were indeed pluripotent. Nonetheless, Yamanaka wanted to go one step further, because if those cells were really pluripotent, then he should be able to use them to contribute to the cells actually in a living mouse. So, ES cells, the other pluripotent cell type we’ve talked about-- if you inject those into a growing mouse blastocyst, they will contribute to the development of the mouse. And they will contribute not just to the embryoid body formation you’re of seeing here, but actually to cells in a mouse as it develops. So the question was, could he use these induced pluripotent fibroblasts-- could he use those to contribute to mouse development in the same way? Well, what this figure shows is in fact that he could. So, here you’re looking at two mouse foetuses. The one on the right is mock-transfected-- so, it didn’t receive the four factors. But the one on the left is a mouse that, as a blastocyst, was injected with those fibroblasts that had been transduced with the four factors-- and also they’d been labelled green so that we could see what happened to them. And what you can see in the fluorescent image over here is that this mouse has had green cells contribute to lots of the different tissues of the body. So, those fibroblasts that had been transduced with the four factors and are putatively pluripotent can indeed contribute to lots of different cell types if you inject them into a mouse blastocyst. So, he was able to confirm that these green cells had indeed contributed to all the different tissues of the body by taking histological sections of mice, like this one. And you can see the stained cells are in lots of different tissues-- so, in the neural tube, in the liver, the heart, the gastrointestinal tract, gonads, and so on and so forth. So, this confirmed that the cells that he’d injected into the blastocyst of this embryo had indeed contributed to all the different tissues of the body. Now, importantly, they contribute also to the germ cells. So, you can see cells here, resident in the gonad. And what that meant was that they contributed-- the fibroblasts that had been injected into this blastocyst contributed to the germ line such that, when mice like this were grown up to adulthood and bred, he was able to produce mice that were entirely derived from these injected fibroblasts. Slide 13 Let me spell that out again. The fibroblasts that have been transduced with the four factors are introduced into the blastocyst at this very, very early stage. The embryo grows up. Some of those blastocysts-- injected fibroblasts contribute to the germ line of this animal, such that when this animal is bred downstream, those germ cells contribute to the next generation of mice. Such that you’re able to generate a whole line of mice derived entirely from those transduced fibroblasts. So, you can see that this is real evidence that those cells-- those skin fibroblasts that have been transduced with those four factors-- really were pluripotent. They weren’t just turning on the reporter gene in vitro. They weren’t just giving rise to differentiated cells in embryoid bodies. They were truly able to contribute to the generation of an animal in exactly the same way as the inner cell Transcripts by 3Playmedia Week 2 © King’s College London 2019 6. mass cells would. So, Yamanaka had really found the biological basis for pluripotency. He’d found the four factors that were both necessary and sufficient to generate pluripotent cells, starting from just common-orgarden fibroblasts. Slide 14 So, Yamanaka reported the work that I’ve just told you about in 2006. In 2007, he published a paper showing that you could do essentially exactly the same thing, starting with human cells. So, the original publication had been with mouse fibroblasts. The following year, he was able to show exactly the same process starting with human fibroblasts. Now, in the years since 2006, 2007, a number of changes have been made to the protocol. It’s been made slightly more sophisticated. We can deliver the genes in slightly different, slightly better ways. But, in essence, the procedure that Yamanaka demonstrated to us has now been broadly adopted, really, worldwide. So, many, many labs across the world now are generating these induced pluripotent stem cells-- these iPS cells, as Yamanaka christened them-- by using essentially his protocol. So, one of the various things we’ve learned during that period is that Yamanaka started with skin fibroblasts. In fact, you can start with essentially any cell type in the body. So, for example, several groups now have shown that you can make iPS cells starting from blood cells. A couple of colleagues that I know about have made iPS cells starting simply with the cells that you’re able to centrifuge out of urine-- so, sloughed off bladder cells that can be isolated from urine. In our lab and a number of other labs, we’ve started from a slightly different sample. So, we start with hair. And the reason for that is we’re quite interested-- as I’m going to go on to tell you about a little later-- in disorders of childhood. So, we’re interested to be able to collect biological samples from children. And we weren’t really keen on trying to take skin biopsies, or even blood, from autistic children. But what we can take really quite easily are hair samples. So, we pluck a hair, or a small number of scalp hairs, just from the head of a child. And from the bulb at the end of the hair, we can grow a population of cells-- of so-called hair keratinocytes. We can then use Yamanaka’s four factors-- engineered now into a different kind of vector, but fundamentally the same as the way he did it-- and from these keratinocytes, we can grow colonies of iPS cells. We can grow these up, expand them, freeze them down, and generate really enormous quantities of these iPS cells. And they’re the substrate for the experiments that I’m going to go on and tell you about in the next section. Transcripts by 3Playmedia Week 2 © King’s College London 2019 7. Module: Biological Foundations of Mental Health Week 2 Building blocks of the brain Topic 3 Exploring mental health using stem cells: What are iPSCs? - part 2 of 3 Professor Jack Price Department of Basic and Clinical Neuroscience Lecture transcript Slide 3 OK, in the previous session, we learned how Shinya Yamanaka and his colleagues taught us how to make IPS cells, Induced Pluripotent Stem cells, starting really from any tissue sample. And I explained how in our laboratory, we start from hair samples, but you can start from blood, or skin, and probably any tissue-- and how using those, we make these IPS colonies. From the IPS colonies, we make IPS lines. So in this session, I wanted to explain what you can do with them, particularly what you can do with them if, like me, you’re interested in brain development, and you are interested in disorders in brain development. Slide 4 We’re obviously interested in nerve cells. So the first job for us is to see if we can take these pluripotent cells, which by definition, as you’ve already heard, can make all the different cell types that make up the body. Can we turn them into neural cells? And, in fact, we can. So what we’re able to do by inducing the cells to adopt a neural fate is get them to make neural progenitor cells. And from neural progenitor cells, to go on and make neurons. Slide 5 So let’s look at this process in just a little bit more detail. So we start with this IPS cell line. We’ve made many IPS lines, and many are available now worldwide. And the first job is to induce them to make neuroepithelium, rather than anything else. We’ve learned to do that by inhibiting what are called the Smad signalling pathways. And the advantage of that is that we’re able to make use of a lot of embryology that we’ve understood now for really quite a few years. So we know from studies principally of mice, but also of other organisms, precisely what it is at the early stages of normal development that push the pluripotent cells, those inner cell mast cells, to go on and give different tissue types. And we know that if we inhibit the Smad pathways that are normally driven by a class of morphogens called BMPs, Bone Morphogenic Proteins, then we can induce neuroepithelium. And sure enough, that’s what happens. So the first step in our procedure is to add what we call Smad inhibitors to the culture of these induced pluripotent cells. Transcripts by 3Playmedia Week 2 © King’s College London 2019 1. And if we culture those cells, we then start to see these neural progenitors. And the first thing to notice about them that makes them really quite distinct from neural stem cells that we’ve come across in other contexts is that they really can do tissue histogenesis. That’s something I’m going to have more to say about in a couple of minutes. But notice how they make proper polarised neuroepithelium. What these cells are trying to do here is essentially trying to make a neural tube in two dimensions. So this is a two-dimensional culture, so a monolayer culture, if you will. But nonetheless, the cells are rounding up to make these what we call neural rosettes, these sort of flower-shaped structures, where they’ve got an actual apical centre just as the apical centre is in the neural tube surrounded by the basal processes of these neuroepithelium cells. So these neural progenitor cells will go on and make neurons. And if you treat them appropriately, you can get them to form post-mitotic neurons. And these post-mitotic neurons will become increasingly mature. And over days and weeks, they will give rise to mature, physiologically-active neurons. Now, one of the things to point out at this juncture is that this process is really quite slow, which in one sense, is a bit of a nuisance, of course. From an experimental point of view, it takes a long time to start from the immature IPS cells, pluripotent IPS cells, and get all the way out to even halfway mature neurons. So this is the sort of timing of this progression in days from day one, for example, to day 35 or 50 out here. But of course, that’s reassuring, because our human development is slow compared to the kind of experimental animals we’re normally used to dealing with such as rodents. So this system really does reproduce the timing and the differentiation processes that seem to underly human neural development. Slide 6 And this just gives you an example of what you end up with. So this is a plate of, in this case, cerebral cortical neurons. And it’s really remarkable. So there we are. We’ve got to plate of, I don’t know, a billion cortical neurons. Just think how difficult it would be normally to get hold of a plate of a billion human cortical neurons on which you could do experimentation. But we can get a billion today, and then I can get a billion tomorrow exactly the same. So it really is an enabling technology that finally allows you to look at neural development in a culture dish-- proper human neural development. Slide 7 Now, I do want to pick up on this point about histogenesis. So I’ve pointed out that these cells really do, when they neuralise, take on a neurepithelial structure. And they really do make a proper polarised neuroepithelium. And if you allowed that to develop, the cells really do try and undergo proper histogenesis. So you can see that down here if we look at this lower picture down here. So here’s one of those neural rosettes that I was telling you about a little bit earlier-- these polarised neuroepithelial cells with their apical surface towards the centre and their basal surface towards the outside. And these cells have been making neurons for a couple of days. And the neurons are the cells stained red. And you can immediately see in this example that the neurons have been generated by the neuroepithelial cells. And just as in vivo, the cells have migrated out to come alive outside the neuroepithelium. And this is an example of a little bit more advanced. And you can see there the Transcripts by 3Playmedia Week 2 © King’s College London 2019 2. apical surface of the neuroepithelium, here are the neuroepithelial cells. And out here the neurons starting to form a lamina in what would be the basal surface of the neuroepithelium. And you can let this go further and further, and this is a much more advanced structure. And you can see the collection of neurons out here just as you would normally see them in vivo in a developing cortical plate. So the cells really have a capacity for histogenesis. That other population of neural stem cells that you might have come across in a different context other such populations simply do not have. Slide 8 And you can push this really a long way. So this is a picture taken from this publication of Madeline Lancaster’s in 2013. What she’s shown is that if you grow the cells appropriately in aggregate cultures, you can actually make them make sort of mini brains-- cerebral organoids, as she calls them. And you can see that these organoids have got real structure. It’s a cortical structure out here developed entirely from these induced pluripotent stem cells growing in a culture dish. So these cells have a capacity for histogenesis that really is remarkable. They really do try and build the nervous system just in a culture. Now, eventually, this process ceases, and it can only go so far, because obviously, a brain can only get so large before it really does become dependent on a blood supply. And there’s no blood supply in these cultures. So there are enormous challenges to carrying this forward infinitely. Nonetheless, these cells have a capacity for histogenesis we’ve really never seen before in other neural developing systems in vitro. And the potential use of that for regenerative medicine is one that I’m not going to talk anymore about today, but it’s fairly obvious. Transcripts by 3Playmedia Week 2 © King’s College London 2019 3. Module: Biological Foundations of Mental Health Week 2 Building blocks of the brain Topic 3 Exploring mental health using stem cells: What are iPSCs? - part 3 of 3 Professor Jack Price Department of Basic and Clinical Neuroscience Lecture transcript Slide 3 So this is the final session in this series about iPS cells-- induced pluripotent stem cells-- and how we can generate them and use them as cellular models. At the start of the session, I suggested to you a thought experiment. And the thought experiment was this-- wouldn’t it be nice if we could reach back in development and study how developmental disorders, like autism, actually arose in the brain of children in utero? Who are going to go on and get a disorder? I suggested that we could use these cells that I’ve been telling you about-- these iPS cells-- to do precisely that. So in this final section, I want to ask and address the question-- how can we use these cellular models to study the aetiology of a disorder like autism? Slide 4 So this is a slide I’ve already shared with you. And it describes the developmental profile that we get from iPS cells as we try and turn them into nerve cells. And I’ve told you how we start with iPS cells. We neuralise them to turn them into neural progenitor cells, and then we slowly, but surely, differentiate them to young neurons and, later, mature neurons. Slide 5 So how can we use cultures like this to start to address the questions around the aetiology of neurodevelopmental disorders? The first thing we can do is fairly obvious. We can take-- we can make iPS lines from lots of different individuals-- both from cohorts of patients that have got particular disabilities or particular neurovariants and from controls-- so from neurotypical individuals. We can grow them in the way I’ve described to you-- making neurons. And we can compare. And we can ask-can we see any differences in the way the patient minds grow from the way-- the way the control minds grow? So that’s one way we can approach disease aetiology using iPS cells. A second way would be to induce mutations in the iPS cells. So you’ll be aware there are a number of ways now of inducing genetic variation-- so genome editing as it’s usually called-- into cells in vitro. So the CRISPR-cas9 system is probably the most popular at the present time. But there are others, like ZNFs and TALENs, that allow you to do similar things. So we could do that. And we know that a number of different genetic variants are associated with different disorders. So we can induce precisely those variants into the iPS cells and ask what difference does it make now to how they develop into the type of neurons they give rise to? Transcripts by 3Playmedia Week 2 © King’s College London 2019 1. A third thing we could do would be to study environmental risk factors. So, for example, we know that in autism, there’s a big increase in risk of a mum giving rise to a child who goes on to get autism if she suffers from influenza during her first trimester. And we think we understand that that’s induced by cytokines-- pro-inflammatory cytokines-- produced by the mother in response to the viral infection. And those cytokines are able to exert an influence across the placenta on the development of the foetus. Well, in principle, we can do that-- we can study that. We can ask if we expose these iPS cells to similar cytokines, what difference does it make to their development? And can we see anything in that difference that we recognise as being possibly part of the aetiology of the disease? Slide 6 So those are just three examples of the kind of studies that we could undertake. So let me ask-well, what kind of phenotypes might you expect to see? In other words, what kind of assays are we going to run on these cells to detect differences? So one obvious thing we can do is look at gene expression. So we can take lines from patients or controls, or we can induce mutations, or we can expose the cells to environmental risk factors, and then ask-- what difference does that make to the expression of genes as the cells develop, both at the earlier stages of neuro development-- like these neural rosettes-- or at the later stages of young neurons and more mature neurons? We can also obviously look at physiology. So I’ve told you that these cells eventually become physiologically active-- so electrophysiologically active. They develop the kind of channels and the kind of receptors that you would normally expect to see in human neurons. Well, we can ask-- do any-- in any of these types of studies, do we see any difference in the types of electrophysiological properties that the cells develop? And a third difference we might look for might be what we might call morphogenetic difference. In other words, I talked about the histogenesis that happens through this developmental profile. Well, is that histogenesis altered in cells from patients versus controls? Do the cells grow a same size and shape as they should? Do they form neurons in the same way as they should? Do those neurons start to wire up and form the appropriate structures as they would in normal circumstances? So there’s an example of some of the studies that we can do and some of the possible phenotypes we might see. Slide 7 So I want to finish this series of presentations by sharing with you just one example. So there are a number of published examples out there now, and this is one paper where they used iPS-derived neurons to study the pathophysiology of a disorder called Timothy syndrome. Now, I don’t want to talk about Timothy syndrome in any great detail, except to say that Timothy syndrome is known to be caused by a particular mutation in a particular gene. And the gene in question is this gene we-C-A-C-N-A-1-C. We tend to refer to it as CACNA1C. And this gene encodes a calcium channel-- this particular calcium channel. And the gene encodes the alpha-one subunit of this calcium channel. And we know from a whole series of other studies that calcium channels are very, very important in signal transduction in neurons, but also in other cells, in fact. So in this paper, the authors generated iPS cells from patients with Timothy syndrome and also from other individuals-- control individuals-- who didn’t have Timothy syndrome-- didn’t have this mutation in the CACNA1C gene. And then they differentiated the iPS cells into neurons using a procedure quite similar to the one that I’ve described to you. And then they asked the question that I posed earlier-- namely, can we see any differences in the neurons that have been derived from the Timothy syndrome patients versus those derived from controlled individuals? And the first thing to look for, obviously, was can they see any difference in the behaviour of the calcium channel? And they found that they could. So what you’re looking at here now is calcium flux in either control cells or cells derived from the Transcripts by 3Playmedia Week 2 © King’s College London 2019 2. Timothy syndrome individuals. And you can immediately see there’s a difference associated with this calcium channel mutation. And it clearly is the calcium channel because if you treat with this inhibitor of calcium flux-- inhibitor of the calcium channel you rectify it. So this is very, very reassuring in the sense that these iPS cells show a phenotype that’s precisely what you would predict, given that these cells carry this mutation of the calcium channel that we know is associated with the disease. So that’s good and that’s very reassuring. But at the same sense, it’s slightly predictable. We’ve taken cells here that have got a mutation in the calcium channel-and, lo and behold, if you look at that calcium channel, it behaves in a mutated fashion. So you could say it is no surprise here-- this is just telling you precisely what you would have predicted. Slide 8 But in this paper, they go and use these cells to do something a bit further. So what they look at here is histogenesis-- this process of building tissue that, as I’ve said, the iPS cells are really very good at. So what they tried to generate was cortical neurons-- cerebral cortical neurons. So let me just use this histological section over here to remind you of some features of cortical histogenesis. So this is a piece-- a section through a human cerebral cortex. This is the pial surface out here. And this would be the ventricular zone. And here is the white matter and here is the grey matter. What you can immediately see from this image-- what, of course, you already know-- is that the cerebral cortex is a very strongly laminated structure. You can see the layers very, very clearly within this piece of cortical tissue. So in-- these iPS cells-- these differentiated iPS cells-- they-- these authors use different markers to try and ask have they got the normal distribution of either upper-layer cortical neurons, like these layer two-three cells here, compared to what are called infragranular cortical neurons-- these deeper layer five-six neurons. Slide 9 And using different markers that label different neurons of different types, they are able to show that, in the Timothy syndrome cells-- here shown in red-- there were fewer cells labelled with the markers for lower neurons. And more of the cells labelled with markers indicating they were supragranular-so the upper-layer neurons. So in other words, the neurons derived from the Timothy syndrome iPSCs had a greater propensity to make upper-layer neurons, and a reduced propensity to make lower-layer neurons. Now when the authors looked at this in a bit more detail, what they discovered is, within this lowerlayer neuronal population, there was another disturbance. Namely, that a smaller proportion of the lower-layer cells showed expression of gene SATB2. Now that rung bells. That seemed important, because we know that these SATB2-positive cells, amongst these lower-layer neurons, are a particular type of neuron. So by and large, these lower-layer cortical neurons can take on either one of two fates. They either become subcortically projecting neurons. Or they become what we call callosal projecting neurons. Now the subcortical projecting neurons project, as the name suggests, to other regions of the brain-the so-called subcortical regions. And those could be the thalamus, the striatum, the cerebellum, the spinal cord. But the SATB2-positive cells belong to the other population. They belong to the callosal population. And the callosal population get their name from the fact that they project across the corpus callosum to the cerebral cortex on the other hemisphere. So what this observation says is that neurons from the Timothy syndrome patients have a lower proportion of the SATB2-positive cells-- that is, a lower proportion of the callosal projecting neurons. Transcripts by 3Playmedia Week 2 © King’s College London 2019 3. Slide 10 Now what the authors were also able to do is look at transgenic mice. And these were transgenic mice that had been engineered to carry precisely the mutation that is found in Timothy syndrome. So these are mice with the same Timothy syndrome mutation. And when they look at the cortical structure in these mice, what they find is exactly the same thing as is being observed here. Namely, that the number of SATB2-positive cells in the lower layer of the cortex of the mouse is fewer in the Timothy syndrome mutated mouse than in the control mice. They see exactly the same histogenic phenotype in the mice as they see in the cells that carry the calcium channel mutation. Slide 11 So what do we make of that? Let’s summarise what we’ve just seen. So what we’re looking at here is cells that carry a mutation in this CACNA1C gene and comparing them with cells that don’t have that mutation. What we see is that the cells that have the CACNA1C mutation have a calcium channel deficit. Now that’s reassuring, but it’s not surprising-- the CACNA1C is, after all, a calcium channel protein. But what we’ve seen beyond that is that those cells have an altered expression in the deep cortical neuron population of this sacB2 gene. And that has led in the mice to an altered callosal projection. In other words, an altered histogenic phenotype. So the development of the cortex seems to be quite different in these cells. So this is an example of where we’ve been able to use iPS cells derived from patients and ask some very fundamental questions about the impact that the mutation associated with their disease on brain development. Slide 12 Let me summarise where I think we are then. So what I’ve told you is that we can use these cellular models to study neurodevelopmental disorders. What I want to suggest to you is that these models that I’ve described have some real advantages compared to other ways that we might study such disease. But I also want to tell you that they’ve got some disadvantages. So this is a balance. So, amongst the pros-- amongst the advantages-- the first thing to say is obviously these are human. So we’re used to having to study neural development in animal models. But what these cells are are actually true human cells. And I showed you that plate earlier, filled with cortical neurons, and pointed out that this was an enormous advantage that we’ve never had before-- the ability to actually study human neurons developing in a culture dish. So one advantage is they’re human. The second is they have what we call good construct validity. And that was-- a good example was the Timothy syndrome paper I cited to you. These cells have precisely the mutation that we find in patients. Of course they do-- they’re actually derived from those patients. And so the mutation that drives the disease-- precisely that mutation-- is there available in the iPS cells. So this was what we would describe as good construct validity for the disease. And that’s one of the assets of this model. Another point I would make is that we’ve got good controls. So we’re able to compare the development of the cells with the mutation with cells derived from individuals that don’t have the mutation. So we’re very well controlled for that mutation in the analysis. I should say that we can go beyond that. I pointed out that we can engineer these cells-- so we can either introduce mutations into cells or, indeed, we can use CASPR/Cas9-type genome editing to remove mutations from cells. And that way we can actually use same cells with the same genetic background with or without particular genetic variants. The system, I would argue, is very tractable. So these are not the easiest cells to grow in culture. But, nonetheless, they are cells that one can readily culture. And that makes this a very approachable system that can be used-- as it currently is being used worldwide-- to study these developmental processes. Transcripts by 3Playmedia Week 2 © King’s College London 2019 4. There is an enormous interest in the pharmaceutical industry in these models, because they seem like they would be amenable to high throughput screening. So we’ll be able to put these cells into drug discovery assays and use them to discover novel drug targets, novel therapeutics for some of the disorders that we’ve been discussing. And the cells that are genetically and phenotypically manipulable in the way that I’ve described to you. Slide 13 So those are the advantages of this cellular system. Nonetheless, it’s important to point out there are some problems that we’ve yet to totally overcome-- some limitations to the system. The first I point is the variability. So I’ve pointed out that we can take cells from different individuals, and compare their performance and the kind of assays that we’ve been talking about. But, of course, one of the problems is that no two individuals are the same, either genetically or epigenetically. And so, if we just took cells from maybe half a dozen different control individuals-- so neurotypical individuals who didn’t have any neurodevelopmental diagnosis at all-- we’d nonetheless find some degree of variability between them. And that variability would be more than we’d expect to see in similar cells taken from, say, mice. Where we’re able to control the genome and we know that every mouse in a colony is genetically identical-- something that obviously isn’t true about individual humans. The second problem is a more pervasive one. And that is that the disorders that we’re studying generally are going to be disorders of the system properties of the brain. What do I mean by that? What I mean is that a disorder, like schizophrenia or autism or ADHD, isn’t just going to be a property of a single population of neurons-- let alone a single set of molecules within a single population of neurons. Rather they’re going to be disorders of properties that emerge from the brain working as a whole. And those properties are currently going to be inaccessible to us. So one of the challenges with the iPS system is to try and build models that will actually have system properties inbuilt. I showed you the beautiful picture of Madeline Lancaster’s where she’s able to grow the cerebral organoids. And I think we’re going to have to do more to try and push those forward-- to try and get us a whole brain, if you will, or as close to whole brain as we can to start to understand how phenotypic differences exist in that sort of gross level. I pointed out that development of iPS cells into neurons is reassuringly slow. It’s good that it’s slow, because human development is slow. But it’s infuriating that it’s slow if you want to get on and do experiments. There’s no question that the fact that you have to wait so long for your cells to develop is a disadvantage in a practical logistic sense. And then the final point is really an important one. Most of the disorders-- in fact, all the disorders I’ve been telling you about are really characterised by altered behaviour. These are behavioural phenotypes. An autistic child is defined as being autistic purely on the basis of clinically observed behavioural differences. Now our problem is these cells don’t behave-- or at least they don’t show the kind of behaviour that we’d be interested in getting from patients. And so it’s going to remain an article of faith the extent to which the cellular and molecular phenotypes that we start to observe-and that I’ve told you about-- actually relate to actual human behaviour. So we’re going to continue to require good clinical phenotyping, good clinical data. We’re going to continue to require animal studies. Mice might not be human, but they do at least have observable behaviour that you can try and relate to human behaviour. So although this is a new model of neurodevelopmental disorders, I think it’s got tremendous utility. It’s going to be a long time before it completely supersedes the other behavioral-based assays and behavioral-based systems that we’re going to need access to. So thank you for listening. That’s my presentation-- or series of presentations-- on cellular models of neurodevelopment. I hope it was enjoyable. Transcripts by 3Playmedia Week 2 © King’s College London 2019 5.