9UNP0135SD Sean Palacek_WordPress

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- Today's speaker for DART talks will be Dr. Sean Palacek, who works with the Chemical and Biological Engineering Department. He'll be speaking on tissue engineering, specifically skin for regenerative medicine. 
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- As much of a discussion as possible to keep me from just talking and you from sleeping. So please feel free to jump in with questions, comments, your thoughts at any particular time. Before I get started, though, I would like to ask how many people here are engineers. Just a couple of us. 

So one of the goals of what I want to do this evening is give you a little bit of an insight into how engineers think about stem cells and regenerative medicine. I'm going to talk a bit about some of the work in our lab that's trying to generate skin from embryonic stem cells. These approaches really can be generalized to a large number of cell types that are of therapeutic interest. The topics we're going to discuss, first a little bit of an introduction into what is an embryonic stem cell; why are these of interest to regenerative medicine; and really, why have these cells rejuvenated the field of tissue engineering. Tissue engineering went through a big downturn in the 1990s. A lot of the money that went into the field didn't end up in clinical products. And investment declined dramatically. This money has started to flow back in the field because of the potential use of stem cells. Then we're going to talk about embryonic stem cell self-renewal and differentiation, ways that we can design culture systems that let us grow up the large numbers of cells that we would need for regenerative medicine applications. And finally, we'll discuss how we can use stem cells in therapeutic applications, specifically, skin tissue engineering. 

The first human embryonic stem cells were derived on campus here. I think most of us know this, in Jamie Thomson's lab in 1998. I'd like to go through this process so we can better understand what these cells are capable of. Most of the current embryonic stem cell lines are generated by the process of in vitro fertilization. So you mix a sperm and an egg in a dish. You grow it for about five days. It grows to the blastocyst stage. Here we have on the order of a hundred cells. There's an inner cell mass surrounded by a trifectaderm. This is before implantation of blastocysts into the uterine wall would occur in vitro. 

The inner cell mass is isolated and the cells are then cultured in a dish on fibroblast feeders. Most of the cells die, but occasionally you'll get a cell that survives and proliferates. These form the embryonic stem cell line. These embryonic stem cells are not quite identical to the inner cell mass. There are some differences in function and gene transcription. But what's important to note is that the embryonic stem cells are really the earliest cell type that we can easily study. Here's a picture of the first embryonic stem cell line. These cells grow in tightly packed clusters. They really like to have cell-cell contact. So if you isolate a single cell, they don't survive very well. 

Although the power of these cells comes from the fact that they can differentiate to any cell type in the adult. So things like pancreatic cells are of interest for treating diabetes. Cardiac cells for heart disease. And neural cells for a variety of nerve disorders. A way we can get cells to differentiate is just by pulling them off a plate and culturing them in suspension, if we remove of the self-renewal factors. So you get these spheres of cells called embryoid bodies. You can think of this as recapitulating some of the very early developmental stages, but it's in a very uncontrolled manner. We don't see the structure form that gives rise to the human body. You do see clumps of different cell types arise in a seemingly random pattern. So what we'd like to be able to do is control this differentiation. 

Before we talk about that, I want to address this idea of the difference between embryonic and adult stem cells. So embryonic stem cells have certain advantages over adult stem cells. Adult stem cells are found in a lot of tissue. They can be harvested and used in therapeutic applications. And adult stem cells are currently being used for therapeutics. I think embryonics have certain advantages that may give them the leg up in the long run. So embryonic stem cells have an enhanced proliferative capacity. We can take a single cell line and theoretically culture it an infinite number of times. They do change upon passaging, however, so there is some effective limit. Whereas adult stem cells, like the cells in our body, have a limited number of divisions. 

There's a difference in differentiation, as well. Embryonic stem cells can form any cell type found in the body, whereas adult stem cells form cell types related to the tissue which they're identified with. So from an engineering point of view, the embryonic stem cells give us a much more scalable processing uniform starting material. We can, in theory, generate an unlimited supply of any tissue type found in the body from a single cell source, whereas adult stem cells, there's a limited availability, not very many people want to donate their brains to harvest the stem cell. And because of the expansion potential here, you can only generate a limited amount of product. We've touched on these properties that give embryonic stem cells a lot of hope, and perhaps some of the hype that's been associated with them. The three main applications for these cells, and one is biological, gives us a window into some of the early developmental processes in humans. We can study this in a dish, whereas it's not ethical and feasible to study it in the body. These cells can be a source of cells and tissues for screening effects of drugs and other compounds on human tissue. Again, it's hard to come up with human heart and brain tissue, but there's a tremendous interest in pharmaceutical and biotech industry in learning how different compounds will affect these tissues. 

So if we can grow them in a dish, they give us a source of cells for the screening. And then there's the regenerative medicine aspect. Embryonic stem cells give us a source of tissues that may be able to be implanted in humans to repair diseased or damaged tissue. There's also a downside to these cells. Because of where they're generated from. Until recently, it required destruction of human embryos to generate these cells. Some exciting work from Jamie Thomson's work here on campus, and a lab in Kyoto, last November, has potentially given us a way to address this problem. What they've been able to do is take adult cells, in both cases, skin fibroblasts, and express four genes in them that are highly expressed in embryonic stem cells. The set of four genes is different in the two studies. But the results are the same. 

These cells return to an embryonic stem cell-like state. So we go from these very flat, large fibroblasts to a small, tightly packed embryonic stem cell. In addition to preventing the destruction of embryos, what this may mean, is that people can generate embryonic stem cell lines from their own cells and tissue. These can then be used for therapeutic applications. So this is really a tremendous breakthrough that the field has been waiting for. There's still a lot of challenges in actually using embryonic stem cells in therapeutics, though. And today, we're going to hit on a couple of these, and not discuss some of the other ones. So one of the problems is that we really don't have very good tools to culture these cells, either at the research scale, or to scale up growth for clinical production. We don't know how to create most cell types from embryonic stem cells. So we have the potential to create an unlimited number amount of tissue from the cells, but we need to learn how to guide differentiation to the desired fate, so we get high purity functional cells. 

Then once we can do that, can we scale up growth and differentiation to get enough cells for use in clinical therapies, and even clinical trials? Some of the other problems that I won't go into today are immunogenicity. Can we generate solid tissues that won't be rejected. And one way to do this may be with patient specific stem cells. And teratomal formation. An unfortunate consequence of implanting these cells, embryonic stem cells, directly into the body is that they can form teratomas, or tumors. Obviously, this is undesired, so we have to make sure that we're not putting undifferentiated cells in with our differentiated desired cells. So the first topic I'd like to discuss, how we can grow these cells and what factors control what happens to these stem cells. So from an engineering perspective, what we're interested in is providing signals to the cells that will either keep them in an undifferentiated state, or allow them to form the cell type we want to make. There are four factors that we're going to discuss. 

One is soluble factors. You can add chemicals to the medium. These will bind to receptors and tell the cell to either stay a stem cell or begin to differentiate. Extra-cellular matrix, so the immobilized protein the cells bind to can guide the differentiation phase. Mechanical cues. I'll talk about this in more detail, along with intercellular contacts, the cells stick to each other and this signals for them to grow, self-renew or differentiate. Here's a schematic showing how we can combine all these different factors to lead to desired outcomes. So we may want to control cell-cell renewal as expand and differentiated cells, differentiate them to the correct phenotype. Other cell processes, we may be interested in acquiescence, when cells stop growing, or even apoptosis, where we can select against certain undesired cells. This raises an issue of the complexity of the environment. 

So what we're drawing the arrows in here suggest that the cells have mechanisms to sense all of these signals in the environment. What we don't know is how the cells process this information. So the context of all these signals, with respect to each other, is critical. Also critical is the temporal nature of how you present these cells. This is not like you have an embryonic stem cell one day and you sort some factors on and the next day you have a beating cardiac cell. This process can take weeks to months. And you're going through many intermediates. The environment that gives rise to say, the first intermediate, may not be the same one that gives rise to the final differentiated cardiac myocyte. We want to be able to dynamically present these signals to the cells to get the desired response. I'd say the vast majority of the field is working in -- factors. 

And I'll talk about that a bit today. I also want to address some of the under-appreciated factors, such as mechanical stress and cell-cell contacts. So biology is really identifying a lot of the pathways and sibling molecules that regulate whether a stem cell decides to stay a stem cell, or whether it wants to become something else. There are several pathways that are indicated here. There's a lot of receptors on the cell surface that bind to growth factors secreted by other cells. And then this will turn on self-signaling pathways to gene transcription that change the cell state. This is not a complete list. And we're just beginning to learn how these pathways interact with each other. Question? 
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- You mention that human embryonic stem cells have the potential to go to differentiate into any tissue of the body. Yet, I haven't heard of, you know, scientists actually making a three-dimensional tissue structure, like a heart or something like that. What are the limitations behind actually seeing published work, as in the first synthesized human heart from these embryonic stem cells? Is it the fact that we don't necessarily know every soluble factor along its differentiation? Or is it just like the three-dimensional molds, something along those lines? 
- So there's a couple problems with that, actually taking the cells and forming a functional organ. The first that you mentioned is getting enough of the desired cell type. And I think that's really what the field is concentrating on now. We can generate cardiac myocytes, which are the little cell type that causes the contraction. 

But I think an even more pressing problem is how we reassemble those. So the cells that we can obtain, whether they're cardiac cells or nerve cells, are randomly oriented and are not connected to each other in the correct spatial organization. That's going to be something that we really need to address. And I'll talk about that in a very basic sense, when we start to talk about the skin. But in a sense, the field has to be able to generate, well, there are two general approaches here. One is to try to generate all the cell types outside the body and then reassemble them onto scaffolds and then implant that. That's going to be very hard. The second approach is to take progenitor cells and put them directly into the body. And hope that the environment in the body will help guide their further differentiation and organization. If that works, it'll be easier. 

But it's not clear that's going to work in all cases. So these pathways primarily respond to chemical growth factors that are in the medium and bind to receptors on the surface or inside the cell. What I want to talk about in a little more detail, are how other signals can affect growth or differentiation. So we're interested in how mechanical forces can stimulate cell differentiation. There are a large number of examples in development, how mechanical forces are required for tissue to properly form. One example is cardiac myocytes, the actual beating activity during heart development is required for your heart to grow properly. Then there are epithelial and endothelial cells that form linings of your body. Your lung epithelia respond to the shear of your breathing, and then the expansion of your alveoli to completely develop. That stimulates their growth and differentiation. Keratinocytes, which form your skin, respond to stretching. 

So your skin is thicker in certain parts of your body, like your knees and your elbows, where they're constantly stretched and mechanically stimulated. Bones and cartilage are stimulated by pressure to strengthen and develop. Also, mesenchymal stem cells can respond to different types of pressure and strain to differentiate to bone and cartilage. So what we decided to do was look at how mechanical forces affect embryonic stem cells. Our initial hypothesis was let's apply forces to stem cells and see if they then form those other cell types that I showed in the last slide. And we're completely wrong. So that's one point one mentions about science. Sometimes what you look for isn't what you see, but it's still going to be interesting. In our study, we grew embryonic stem cells on a flexible silicone rubber membrane substrate here. So we have cell colonies on a flexible silicone membrane. 

And we can apply a vacuum onto that membrane. What that does is stretch the membrane and applies a strain to the membrane. The strain is transmitted to the cells. And we do this in a cyclic manner. So we apply a vacuum, release, apply a vacuum, release. And we can control the magnitude of the strain, as well as the frequency of the strain. What we found is that when you strain the stem cells, it actually inhibits the differentiation. They're more likely to stay a stem cell. Now, one thing I didn't mention, the culture is that if you keep these embryonic stem cells in colonies, and let them grow really large, they begin to spontaneously differentiate. In a typical culture, what you do is every five days or so, you take all of your cells and harvest them and enzymatically or mechanically chop them up and re-plate a certain fraction of them. 

In this case, we allowed the cells to grow for a week, and just let them keep growing. We got these very large colonies and we'd get these areas of differentiation. The cells that don't express this Oct4 and aren't in green, are some cell type. We don't know what they are, but they're no longer embryonic stem cells, whereas these cells here are still stem cells. If we apply strain to those cells, so we're stretching them ten percent, so just elongating them by ten percent longer than their normal length, at ten cycles per minute. So every six seconds, we go through a cycle, we keep them completely undifferentiated. After two weeks, the effect is more dramatic. So cells that aren't exposed to strain will undergo spontaneous differentiation. Almost every cell in this culture is no longer a stem cell. We don't quite know what they are, but we know they've differentiated to some particular lineage. 

Whereas the cells that we apply strain, we have some differentiation, but not very much. We can quantify this. How we do that is label the cells with an antibody that only binds to a protein expressed on the surface of embryonic stem cells. Here, we applied ten percent strain, ten cycles per minute for ten days. And here, two populations. So any cell in this region out here is undifferentiated. The cells that were exposed to strain, primarily undifferentiated. The cells that weren't strained are differentiated. What happens if we drop the strain to eight percent? Surprisingly, it has absolutely no effect. 

So the two strain, non-strained populations are identical. They both have a large number of differentiated cells and a few undifferentiated cells. This is a threshold for them. And we have to be above a certain level of force to keep the cells undifferentiated. Below that level, it's like not even having any force at all. We did quite a bit of characterization here, and I don't want to go too much into the details. But what we're really interested in is how are the cells sensing force and what's it doing to the cells. We looked at a large number of pathways. And it looks like the force is somehow stimulating the TGF-beta pathway. So in embryonic stem cells, TGF-beta is a small protein that binds to receptors on the cell surface. 

Then this turns on a pathway that leads to gene transcription and keeps the cells in an undifferentiated state. Their related protein is called bone morphogenic proteins that stimulate differentiation. A number of other labs have shown that this TGF-beta signaling keeps cells in undifferentiated and pluripotent state. And that they activate this complex called SMAD2/3, which then goes to the nucleus and turns on gene transcription, whereas the BMP activates the SMAD1/5 that promotes differentiation. I won't go into the real experimental details here either, but what we found was that in the cells that we apply the stretch to, they turn on TGF-beta expression by about a factor of ten-fold. So stretching the cells causes them to make TGF-beta. They secrete this into the medium, and it binds to themselves and their neighbors. And then this is an autocrine or paracrine move that keeps the cells in an undifferentiated state. If we add this chemical inhibitor of the signaling, we keep the turnoff signaling through the TGF-beta receptors, and the cells will differentiate. We also just looked at signaling activity. 

If we look at the level of the signal downstream of TGF-beta receptor, the strain turns on this SMAD. If we don't have strain, we have very little phosphorylated SMAD, and we can inhibit it with this inhibitor. So the signaling through TGF-beta directly correlates with the cell state. We think this is an example of how cells sense mechanical signals. They sense chemical factors, and then somehow they process this information to keep them in an undifferentiated or differentiated state. The other aspect I'd like to talk to you about in terms of cell culture engineering is how do we control cell-cell interaction. The colony I showed earlier, the small picture here, these colonies grow as three-dimensional clumps. There's a layer, and it grows hemispherical clumps of all different sizes and shapes. There's a lot of anecdotes in the field about how small colonies may be better for a particular lineage, and big colonies may give you a different lineage. But there have been very few studies that really indicate why or methods that give us methods to actually control this. 

One of the first studies that was published in 2005, what that lab did was quantify the size of clumps that give rise to two types of blood cells. The myeloid lineage and the erythroid lineage. The way they do this they take a plate of cells and enzymatically dissociate these into single cells. And they count cells, put them in a small tube and centrifuge them, just spin them together into a clump. Put these on a plate, expose them to growth factors that can either stimulate myeloid or erythroid growth. They found that for the erythroid lineage, there's an optimum cell number, whereas for the myeloid lineage, as long as you're above a certain threshold, you get a lot of differentiation. This method is very labor intensive. And it's somewhat artificial. Because as you force these cells together into a clump, they tend to like to disaggregate. So we decided to try to develop a method that really lets us control the size and shape of these colonies as they grow. 

This gets us into some of the materials engineering world. So what we want to do is form these surfaces, they're called microwells. These little three-dimensional wells that we can put cells in and they will fill up the wells to form an aggregate of a certain size and shape. We create a rubber stamp here using a silicone wafer mold that's manufactured somewhat like you would manufacture a computer chip, although it's much simpler than that. Then this rubber stamp forms a template for polymer molds that we're going to use to make the microwell. In this area between the spacer and the stamp, we inject a liquid polyurethane. This gives us this microwell shape. What we want is for cells to be able to attach and grow inside the well, but not the area between the well. We're going to treat this area outside the well with a compound that keeps cells from sticking. We deposit gold at an angle. 

Gold is vaporized and deposited at an oblique or steep angle, like this. The gold will hit the area between the wells, but will be shaded from the bottom and side, because we're coming at this angle, it can't hit the bottom. Then we're going to react a molecule with the gold. This molecule is going to keep the cells and proteins from sticking. So this molecule, one end will bond to the gold, and the other end is a trophyline glycol. The trophyline glycol inhibits protein cell attachment. When we add protein, it can only attach to the bare polyurethane in the bottom. We add cells, they'll attach to the protein and grow to fill these. What we're left with is somewhat like an ice cube tray, but all of the trays are filled with cells. Here's a phase contrast image of what these wells look like. 

These are 200 microns on each side, and about 100 microns deep. So these wells will hold, on the order of 1 to 5,000 cells. If we incubate with protein, the protein only goes inside the well. Then we add cells and the cells will grow to fill up the well. These areas in here, what you see are bubbles that are under the surface of the polyurethane. So if we do a stain for dye, you'll only see cells inside these wells. What's going to happen is we put in a relatively small number of cells. They'll bind and start growing. The cells will grow in three dimensions and fill up the well. So here you can see, the differences in color are because you have different levels of growth. 

Here we have just a couple layers. And this dark color indicates that we're growing above and outside of the well. You can see the number of cells here are larger. The Calcein AM just indicates that these cells are actually alive. The cells have been cultured for 19 days without passaging. At this point, they mostly filled up these wells. The cells remain proliferative. Even as they fill up the wells, they keep dividing. What they're going to do is grow up outside the well and form these little mushroom type caps that will eventually break off into the medium. So a side view, cutting across the well and showing the colony is provided here. 

The green shows the cells are undifferentiated. The nuclei are shown in red. This sample has been somewhat mangled, because we took a razor blade and chopped it up. Eventually what will happen is the colony will fill up the well. It will grow up above. And as we add medium or change the medium every day, it's just going to shear that off. So what we're left with is a full well of cells. Eventually, we want to be able to pull the cells out of these wells and then use them, either in growth or differentiation type experiments. We can do that enzymatically, if we add an enzyme that's going to break the contacts between the cells and the substrate. What we're left with are these little cube in suspension. 

These are all of the same size and shape. Once you get them to differentiate, you culture these in suspension to keep them from clumping together. Overnight, they're going to switch from this cube shape to a spherical shape. One of the things that we didn't expect is that when we culture cells in small wells, they remain almost completely undifferentiated. So this is somewhat analogous to what we saw with mechanical strain. When we stretched cells, they don't undergo spontaneous differentiation. Also, if we keep the colonies fairly small, it keeps them differentiating. We don't really know why yet. One theory is that just as the colonies get very large, they may secrete factors that once you get to a critical concentration, cause differentiation. I think where this technology is most useful is that it lets us get uniform populations of stem cells. 

The size distribution of an unconstrained, or just a typical stem cell culture is shown in these light blue-green bars here. What you get is aggregates ranging from 160 to greater than 500 microns in diameter. Using microwells, we can create these five different completely dispersed populations using different size features. This is a way to make the culture much more uniform, which then lets us control the environment. What we're interested in doing now is looking at how this initial starting size of the colony affects what cell type we get. So by culturing the cells in suspension, we get this much more homogeneous distribution in sizes and when you compare it to the standard embryoid body method. We're working with a number of people on campus here and around the country to look at specific lineages. So what size of embryoid body gives is the most myocytes, for example, insulin secreting cells or neural cells. So just to talk about the self-renewal aspect, to summarize that a bit before I move on to tissue engineering. A large number of labs around the world have identified soluble factors that keep cells in the undifferentiated state. 

The message that we're trying to get across is that it's not only soluble factors that are important. But that we can keep cells undifferentiated by applying mechanical forces to them. And this appears to work by stimulating TGF-beta cell signaling. Also, cell size is important. So regulating cell-cell contact can affect differentiation. Keeping cells small and minimizing cell-cell contact will keep them self-renewing. But if you keep them large, you begin to see spontaneous differentiation. So I think I'll stop and take any questions or comments here before we move on to the tissue engineering aspect. Yep? 
- So the mechanical strain, you said, can keep the cells from differentiating, but isn't that just a direct effect of the TGF-beta? 

Couldn't you just add the TGF-beta to keep the cells from differentiating? Is the strain just a more legitimate way to do that, or is there a reason you'd prefer to do it with the strain rather than just adding the growth factor? 
- Good question. So I didn't show all the detailed experimentation here, but adding TGF-beta doesn't completely replicate the effects of strain. It replicates about 75% of it. So we think that's the dominant effect. But not the entire story. Yes, if your goal is to keep cells in self-renewing state, I think the best way to do it, if you can add growth factors is to add growth factors. There's a reason so many people are still doing those types of signals, because they're much easier to manipulate. Trying to scale up how you apply strain is difficult. 

But one of the messages here is that when you are designing a culture system, it's important to keep these things in mind. So other types of forces that are important include shear. So if you're trying to culture these cells in suspension, you can't get around the fact that you're going to apply shear to them. We really need to know what effect that will have on the cell. Other questions? So then let's talk about how we can use these cells in therapeutics. And there are a large number of labs on campus, in the country, around the world, that are trying to generate specific cells for therapeutic applications. One of the ones I want to talk to you about today, not a lot of people are studying. This is epidermal keratinocytes. So, can we make skin cells? 

And I'd like to introduce why we want to use stem cells for tissue engineering, and specifically for skin tissue engineering. The goal of stem cells in regenerative medicine and tissue engineering is to take these very early, primitive human cells and generate the perfect replacement for cells, tissues, and organs that may have been damaged or diseased. There are two main ways to this. I think we talked about it in response to some of the questions before. But either an in vitro method or an in vivo method. In the in vitro method, what we do is take these embryonic stem cells, differentiate them into specific cell types, and assemble these cell types into an organ, and put that functional organ into the body and assume it stays relatively static. This is nice to think about, because it's a very controlled environment. You can control what happens at each differentiation, and you get a fully functional organ that you can put in the body. The problem is it's too complex at this point to really be effective. Then there's the in vivo method, in which we directly place the stem cells, or their precursors, into the body. 

And hope the environment at which you place them will guide their development and differentiation. This is much easier. You can take stem cells or their early progenitors, toss them into a body and see what happens. So even though it's easy, the problem is that it doesn't work very often. If you directly put those stem cells in the body, you'll get teratomas. If you put early progenitors, the environment isn't quite right to stimulate their total development. That makes sense because in the early steps of human development, the environment is different than it is in the adult. So there will probably be some blend of the in vitro or in vivo types of approaches that will work. And that blend will be different for different tissues. A lot of different cells have been shown to be generated from embryonic stem cells. 

This is a bit old, and it's very incomplete. But you get neural cells, skin cells, cornea, cardiac, bone, blood cells, liver cells. So we're beginning to be able to make functional adult cells and embryonic stem cells. I think one of the big questions based in the field is how do we put these together and use them in a therapeutic sense. Just because you can make a neuron doesn't mean you can put it at the right spot of the brain or spinal cord and have it interact with the local environment and be wired properly. Some of the challenges that are facing the field that I think we need to address, as scientists and engineers, include the low yield. The cell types I put up on the last slide, we can make them, but the actual number of each cell type is relatively low and the purity is low. A lot of this relates to the complexity of the environment. So the types of signals you give to cells are going to be important. And it's not just add this one growth factor and you get your desired cell type. 

Defined conditions are going to be important. A lot of the culture methods now use animal-derived products, cultures with other cell types to get these cells and tissues through FDA approval. These add complications because it's hard to say one batch will maybe be the same as the next batch. Also, generating the cell that have the complete, correct phenotype is important. So often, you get some properties of a cell type, but not others. One example, and there's been a lot of excitement in the past week, that insulin producing cells have been generated from embryonic stem cells, and that these can "cure" diabetes in mice. The problem is that these insulin producing cells don't entirely respond to glucose the way they should. So there are certain functions that are missing from these cells that are going to be critical to their use. And finally, how do we assemble different cells into functional tissues? There's a three-dimensional orientation of cells that give tissues and organs their true function. 

This brings us to skin. So why are we studying skin? Well, there's a market for tissue engineered skin products. The two types of patients that would benefit most are burn victims and people with wounds that don't heal. There are about 10,000 deaths per year due to burns. And burn deaths are caused by loss of skin function. People lose the ability to regulate their temperature and the water content in their body. Another million people experience permanent disabilities every year because of burns. So if we can generate replacement skin that would improve the quality of life, or perhaps save lives. Non-healing wounds is another use for skin tissue engineering. 

And in fact, these types of wounds are currently being treated by artificial skin products. The two main types of non-healing wounds are foot ulcers in diabetics and venous leg ulcers. Both of these are caused by a diminished blood flow into the extremities, which then results in low rates of wound healing and diminished immune response. And amputations in diabetics are common, typically in the feet. And venous leg ulcers can also cause amputation. The costs to the healthcare system runs in the billions of dollars because of the number of people that have these types of problems. In trying to engineer skin, what we want to do is look at the natural tissue, figure out what it does and see if we can replicate its structure and function. Or, provide its function through a slightly different structure. The critical aspects of skin to keeping people alive are the mechanical barrier and dessication barrier. You keep micro-organisms out, infections out. 

You keep water in. And some of the current products can do that. Some of the other functions that would be nice to maintain include sensory. Your skin contains nerves that give you the sense of touch. Immune response to fight infections. Thermal regulation. This is often controlled by blood supply. The skin is vascularized, and keeping its vascularization not only keeps the cells alive, but helps regulate flow in the body. Then there are also things like pigmentation, which are little bit more cosmetic. The structure of skin is shown here. 

You have the epidermis, which is the outer-most layer that provides the mechanical dessication barrier. And most of these other functions are present in different cell types in the dermis. We also have sweat glands that involve thermal regulation, hair follicles. It's actually quite a complex organ. The first generation of tissue engineering products really consist of only one or two subtypes that give us an artificial epidermis. The maybe not so current skin substitute, some of these are not currently available. But historical skin substitutes are shown here. The simplest type of artificial skin is really just an artificial membrane. So you have, perhaps, a silicone rubber membrane that's fused to a nylon mesh. This has peptides on it that help the cells move and repopulate the wound. 

You place this over a wound with the silicone rubber membrane facing the exterior. And as the wound heals, the cells will crawl in and repopulate this mesh. At a specific time point, you cut off the silicone rubber membrane. Then there are more biologically based materials. One is collagen based dermal analogs. This is cadaver skin that's been treated to kill living cells. It's just the matrix that you would place down. Then this would allow cells to crawl in and repopulate the wound. These are passive materials that stimulate growth and proliferation of cells from outside the wound into the wound. The later generation skin substitutes have actual living cells in them. 

One of them contains human keratinocytes and fibroblasts in an artificial matrix of extra-cellular proteins. This replicates the dermis and epidermis structure. The problem with this material is that when we implant it into a wound, it's not vascularized. So these cells don't have a blood supply and they don't live very long. What they do is the time that they are there, secrete factors that allow cells to repopulate. So these cells do not become a permanent part of the wound site. Our vision is to be able to completely replicate the structure and function of skin, where you have vascularization, nerves, sweat glands. How we're starting out though, is just to try to get some of the cell types that are found in skin from embryonic stem cells. So what we've been working on is keratinocytes. These cells form epidermis. 

And they stratify into multiple layers. The basal layer of keratinocytes will grow over the dermis. Their role is to form a mono layer of cells that are then going to divide and further differentiate. So as they divide, they move upward. They begin to secrete keratins, which are water insoluble proteins, water resistant proteins. The cells will eventually die and form this barrier at the outside of your skin. Typically, you have ten to 20 cell layers thick of keratinocytes at any point on your skin. About five years ago, a lab at Harvard was the first to demonstrate that embryonic stem cells do form these skin keratinocytes. What they did was take embryoid bodies, or undifferentiated cells and injected these into mice. They harvested the tumor that arose. 

They found cells in the keratinocyte lineage. So cells expressed the markers, P63 and Keratin-14. This indicated that they're roughly equivalent to these cells at the basal layer of these proliferative cells. So this was a very important study. It showed that it's possible to make the keratinocytes from the embryonic stem cell. The problem is this isn't a very effective way of doing it. You have very low yield of these cells. You end up killing a lot of mice. And it doesn't give you much insight as to how these skin cells develop. If you want cells for a therapeutic application, we need to find a better way to do it. 

And our approach is to see if we can induce this factor P63. P63 is expressed in a lot of epithelial cells, so the cells that form linings of virtually every organ in your body. We figured if we can turn on expression of this, it will give us progenitors that are more likely to form skin cells. It won't necessarily give us skin, but it will get us closer along that path. Our first strategy is to use this embryoid body that we discussed earlier. You take your undifferentiated cells that are growing in a dish. And then you pick them off the substrate and culture them in suspension. They begin to differentiate to form a large variety of cell types. The green cells here represent the cells we want, keratinocytes. You plate these on a dish coated with a extra-cellular matrix. 

We happen to use collagen, because that's the main component of skin. What happens, then, is your keratinocytes will migrate out away from the other cells. This makes sense, because their function is in wound healing. They're able to crawl and move, close wounds very fast. So they walk away from these other cell types. We can then somehow isolate these keratinocytes, purify them, and induce differentiation and stratification. Here's an image from that migration phenomenon. We dropped an embryoid body at this point here and then waited, I believe a week or a week and a half in this experiment. So the cells began to crawl out. This panel corresponds to this image. 

So these cells still express Oct-4. They're undifferentiated embryonic stem cells. Whereas the skin progenitors walked away from the undifferentiated cells, and formed these relatively pure regions. If we magnify the region here, we see that they have this epithelial like shape. They tend to form tightly packed sheets of cells. And they also express p63. Question? Scale? These individual cells here are roughly 50 microns wide. So this colony here is on the order of a couple millimeters. 

There are thousands of cells in that particular slide. We did a fairly exhaustive analysis of the different factors that will guide keratinocyte differentiation. I don't want to go into all the detail here, but the soluble media you grow the cells in is important. So we took these embryoid bodies, put them in collagen for 11 days. Then we put them in different commercial media formulations that are used to culture keratinocytes. The idea here is that these commercial media will contain a number of different growth factors that keratinocytes like and this may stimulate their growth. We found that some give us a lot of keratinocytes whereas others are completely ineffective. We're currently trying to figure out what factors are the ones that give us keratinocytes that aren't in the others that don't. The matrix you plate the cells on is important. Here, we have the embryoid bodies cultured for two weeks. 

We put them on different matrices and cultured them for an additional month in the defined certain mediums which we found gives us keratinocytes. If you put the cells in matrigel, which is commonly used to keep cells undifferentiated, we get a very low yield. What happens here is that the cells actually don't differentiate. You still get a lot of undifferentiated stem cells. In serum, we get higher level differentiation. Collagen, about 15%. Interestingly, gelatin gave us better than collagen. So we were able to obtain about 25% of the cells in the culture are keratinocytes. We're not exactly sure what the others are, probably a mix of a variety of cell types. Question? 

- When you're saying we have keratinocytes, are you just referring keratinocytes as cells that express the K14, or whatever or are you going further than that and looking into more detailed expression? 
- Good question. Here, we're seeing keratin-14, and that isn't sufficient to claim that they're actually keratinocytes because certain other tissues will express keratin-14. But later we looked at the ability to terminally differentiate to express other keratinocyte specific proteins. So it's probably not fair of me to say that these are keratinocytes at this point. These are cells that have the potential to form keratinocytes. This is our best result using embryoid bodies, about 25%. The problem with embryoid bodies is that by culturing the cells in suspension in these undefined conditions, you begin to see differentiation toward a lot of different choices. So if we want a pure population, we either have to get rid of all the other cell types or purify the keratinocytes. It's easier, and perhaps more effective, to do a direct differentiation protocol. 

So instead of forming embryoid bodies and letting them differentiate randomly, you take the adherent cells and you treat them with different compounds that direct their differentiation from the start. One of the factors that's commonly used to direct differentiation is retinoic acid. Retinoic acid is a form of vitamin A. It's structure is shown here. It's been shown to be important in skin development, as well as development of other tissues, including neurons. Mice that lack the retinoic acid receptor don't form basal keratinocytes with the same structure as mice that have the retinoic acid receptor. So our hypothesis is that if we apply retinoic acid at the proper dose and time, we can specify for keratinocyte formation in embryonic stem cells. What we do is take these embryoid bodies, these colonies of cells in suspension and apply retinoic acid as we form them, keep them in suspension, and analyze for gene and protein expression after suspension phase, then after plating for an extended period of time. What we noticed is that if you apply retinoic acid, you completely change the nature of what these cells or EB clumps look like. So without retinoic acid, they're typically very densely packed. 

With retinoic acid, they're more clear. Other labs have sectioned these and shown that there's hollow space inside of these cells. What you tend to see is epithelia-like sheets form, and these make up hollow spheres. The gene expression analysis that we perform after either two days in suspension and exposure to retinoic acid over eight days shows that in fact, retinoic acid applied at this time point gives us more expression of markers that are epithelial specific and less neural specific. Oct4 is a marker of undifferentiated cells. And retinoic acid almost completely wipes those out, whereas K18 and p63 here are turned on by retinoic acid. The neural markers Nestin and Pax6 and Sox1, there's no effect at two days, but after eight days, we see a down regulation. So we're getting more epithelia but less neural progenitors. If we plate the cells after ten days, that have been exposed to retinoic acid, we plate them on collagen or gelatin, and grow them keratinocyte growth medium, we can quantify the number of K14 positive cells. The cells that have been exposed to retinoic acid give us more of the keratinocyte progenitors than the cells that haven't. 

Unfortunately, we're still only roughly at this 30% to 40% purity here. What we tend to see are these regions in red that are keratinocyte progenitors surrounded by these other cells types, in this case, neural progenitors. We still have the significant neural progenitors population that we want to get rid of. The important point I want to make here is that it matters when you apply the retinoic acid. If you apply the retinoic acid just earlier in the embryoid body stage, we get a lot of keratinocytes and we knock down the number of neural progenitors. If we apply the retinoic acid, not during the embryoid body, but during the culture, it doesn't give us keratinocytes, but it actually enhances neural formation. If we apply it in both, EB and adherent, we get some upregulation of keratinocytes, but we knock down the neural population. So the optimum time for getting keratinocytes is to give the retinoic acid early, but not late. So this moves us to the direct differentiation aspect. If we don't take the cells and put them into suspension, if we directly add retinoic acid to the embryonic stem cells, we see the same qualitative effects. 

The retinoic acid turns on keratin-specific markers and it keeps the cells, it turns off the self-renewal. But what it does is gives us a much higher purity. So here, we have six days of retinoic acid, and then extended culture. We obtain an almost pure population of keratinocytes, these form a very confluent epithelial sheet. This gets to the last question that was asked. Do these have the potential to truly form skin. The answer now is not yet, but they're getting closer. One of the things that these keratinocyte precursors are supposed to do is stratify into multiple layers. They do that, but only in small islands so that it doesn't occur over the entire surface. One of the ways to stimulate this is by adding calcium. 

If we add calcium, we get stratification and expression in Flaggrin and Involucrin, which are specific to the terminally differentiated cells. They also can form these epithelial sheets. If we detach them from the surface, we can pick this up with a tweezers and it remains intact. What we're currently doing is trying to look at the signals that will stimulate further stratification and differentiation, things like mechanical signals as well as chemical signals. So this is really a work in progress. We're getting closer in getting regenerative cell types. Eventually, we'd like to combine keratinocytes with other skin cell types, endothelial cells, nerve cells, and so forth, to truly regenerate skin. But we're a ways away. So we need substantial improvements in both cell culture design for keratinocytes, as well as hESC-derived cells to be able to use these in regenerative medicine. It's going to be a complex problem, so soluble factors and matrix factors affect the yield. 

Things like retinoic acid and collagen and gelatin here. Mechanical factors may also play a role. And finally, we've been able to show some function here in terms of terminal differentiation and stratification. So before close here, I just want to acknowledge the people that did the actual work here, the experiments were performed by Lin, Christian, Yin, Somen, Jeff and Samira. And this work is really a collaborative effort. The things I've talked about weren't only from my lab. Juan de Pablo is our materials expert. A lot of help from Jamie Thomson on embryonic stem cell culture and advice on how to keep these cells undifferentiated. Lynn Allen-Hoffman in the Pathology Department is a skin expert there. Also Derek Hei, Jeff Jones and Igor Slukvin have been working with us on other aspects growth and differentiating. 

Thanks for your attention and I'd be happy to take any additional questions you may have. Yeah? 
- Hi, Professor. My name is Joe. So, I can see the rationale of your research, just for generating skin layers, especially for patients, also those with diabetes ulcers. But when I thought of those two different, I would say, injuries, I immediately thought about infections. Because in the burn unit, it's very common to have resistance, even MRSA. And the thing is if you actually just cover the wound site with new skin layers, it is possible that you're sealing the infection. Then you can also have bacteria and even worse symptoms in the person. That may even cause the death in burn patients more. 

So I guess my question is that is it possible to just use your model and combine it with another treatment to prevent those bacterial infections? 
- Sure, that's a great point. You would often want to combine, you'd need to combine this type of model, even if in our wildest dreams are realized and you get fully functional skin. Once you put that in, it will take a while to integrate it into the wound site. By that time, you'd still have a lot of problems with infection. So you'd need to treat it with antibiotics and other methods. But I do think that a fully vascularized functional skin that integrates would be vastly superior to these other types of dressings, because you'll get blood flow to the area, and you'd get immune response there. Then you'd also have local dendritic cells that reside and offer some infection fighting potential. But that's many generations away. 
- Thank you. 

- Do you think it would be most advantageous for patient-specific tissues, if you started out with the induced pluripotent stem cells rather than the basic human embryonic stem cells? 
- Definitely. If these induced pluripotent stem cells end up to have all the functions that we need, it'd be much easier to generate a bank of cells. There are a couple ways to envision this working. One is take, if someone needs treatment, you take their cells, generate an IPS line from that, then grow the tissues. This may work for someone who has diabetes who can be treated for an extended period of time, because it takes a long time to generate the cell lines and tissues. It would be less likely for a burn patient who needs something off the shelf. You may have, for tissues like that, for a heart, for example, it would be banks of different cell lines that are compatible with different antigens. So in that case, you have banks. I'm not an immunologist. 

But you'd need hundreds and thousands of lines, and have tissues that have already been made for those. So if you could have an off the shelf product, that would be great. Or if you could just start with a line and a bank and make the cells as needed, that would be another way to go. I think that depends on the needs of the specific patient and disease, which one would be optimal. 
- Hello, I had a question. You mentioned how the diverse or different kinds of cells that you can create. You mentioned how they may not give you full function, such as the insulin cells that were created. But is there any knowledge on the life span and longevity of those cells compared to cells that would normally? 
- One problem that I didn't talk about in our skin work, is that these cells have a much shorter life span than primary cells, and we don't know why. You can get about ten population doublings. 

It still gives you a pretty substantial expansion. But primary cells should have 30 to 40. One of the theories is that there's something in this selection process that's prematurely aging the cells. We don't know what it is. So there's an extensive amount of literature in the aging field, in terms of -- activity, and other types of pathways that regulate how much cell divisions a cell has. If anything, we expected embryonic-derived cells to have a lot of population doubling, since they're coming from a very premature cell. In our case, that doesn't happen. I don't know how general that is to other lineages though. 
- Do we currently have any cell lines that are anywhere close to being like, having full expression? Like as far as you were talking about the K14. 

I'm sorry, I'm struggling how to word this. But basically, do we have any types of cells out there derived from stem cells that, like, that are anywhere further along than skin. 
- It depends. I'm definitely further along than that, but nothing I would want implanted in me. So, there's room for improvement in a lot of different fields. I think what's limiting a number of fields, and insulin secreting cells are pretty far along, neural cells and cardiac cells, because there's a lot of people studying them and there's a big market for treatment. What's limiting us is the ability to integrate them into functional organs. Again, insulin secreting cells may not be as much of an issue. The tissue engineering field from the '80s and '90s has figured out ways of encapsulating these cells and implanting them. They don't necessarily need to be in the pancreas. 

Once they make insulin, as long as it gets in the blood stream, it's fine. But neural and cardiac lineages, even if we get cells that are effective, how are you going to incorporate them? It kind of brings it back to how far along are we. One of the advantages of skin is that it is one of the two or three commercial products that are out there in tissue engineering. So we know a lot about how, once we have the cell type, how we can incorporate that into the current constructs. Cartilage is the only one that there are products. Our rationale for getting into skin isn't necessarily because there's a shortage of artificial skin. You can culture skin from adults and grow as many cells as you want. It's more that this is a good model system for looking at how we can form organs and tissue. While the skin field is a little bit behind, in terms of generating cell types, I think once we have them, we'll be able to move forward faster than the neural or cardiac cells, which are going to have these big problems in how you design the 3-D constructs. 

I hope that kind of gets at what you're asking. 
- Do we, are they doing any like microwave stuff, to see what the expression difference is between say, one cell base versus another, and then seeing how far they are there? 
- That's a good point. The issue of how do you compare the embryonic stem cell derived cell with the primary cell you want to replicate. One way that's commonly used is microwaves to look at gene expression patterns. You'd want them to be as similar as possible. The nice thing about these assays, is that you can, if you see differences, you can get ideas on what's different between the cells. The other type of analysis people will look at is functional tests. So do the cells make insulin? Do they beat properly? 

Do they transmit electrical signals? Ideally, the gene expression relates to the function. As far as I know, there is not a current cell type that is far enough along that it's close to being identical to the primary cell. And the in vitro differentiation process, the environment that surrounds it is very different from what the cells see in people, so it's not surprising that they're not identical. 
- It sounds like two of the problems that are going on right now is that you're trying to get these cell to mimic the primary cells and then also scaling that up. You talked about the challenges. What's going on in terms of getting these scaled to the necessary quantities? 
- Some numbers here, if you want to treat a patient, you would need billions of cells. If you grow the cells in a dish, you can get millions of cells. That's thousands of dishes of cells at a lab scale, which is completely unfeasible. 

That's just to treat one patient. So if you have tens or hundreds of patients in a clinical trial, there's no way you can even culture the cells. Then you multiply on that, there's an efficiency problem. You don't get 100% efficiency, so you're dealing with tens, hundreds of thousands of dishes. I think what the field needs to do is look at what other adherent cell culture researchers have done. So this is primarily work that has been done in protein production. There's very few therapeutic cells out there that have impacted the cell culture community as much as protein production. So here, can we culture the cells, really, in stir tanks and suspension to get us enough cells? That involves removing the need for the matrix or being able to culture cells on microcarriers, which are small spherical particles that you can stir and keep up in suspension. And there are people who claim they've been able to culture cells in these types of configurations for short periods of time, but not to the scales that we're talking. 

So to be able to move on, even to clinical trials, there has to be some engineering of scale here. That's something that's hard to get. There's a chicken and egg argument here. People aren't interested in the problem until it's time to do the clinical trial. But then they'll be saying, why didn't we work on this problem in the mean time? My idea here is that we should assume that something is going to come out of stem cells. We may not necessarily want to work on scaling a particular lineage yet, but if we can at least learn how we can keep the cells growing in an undifferentiated state and expand those, it will pay dividends down the road. I thank everybody for your attention. 
(applause)