9UNP0159SD Gary Lyons_WordPress

- Thanks for coming today to our PLATO Frontiers in Life Sciences presentation. Today's speaker is Gary Lyons. He's with the Department of Anatomy here at UW-Madison. And he's also with the new Stem Cell and Regenerative Medicine Center, which started just recently. He'll be talking about heart disease and cardiac stem cells. Please join me in welcoming Gary Lyons to the PLATO series. 
(applause)
 
- It's a pleasure to be here. I'm glad Tom was actually outside and helped me find where this is. 

Otherwise, I'd probably still be wandering around, beginning to get very worried. So I'm a professor in Anatomy in the School of Medicine and Public Health. I'm not an MD, so I cannot answer any of your clinical questions. But I know quite a bit about the heart, less about heart disease. And what we study is stem cells, and how those stem cells might be useful in current studies that are ongoing clinical trials. And also, we'll discuss a little bit about what are the future possibilities for using stem cells to help regenerate the heart. So you may be well aware that heart disease is the leading killer in the U.S., and in fact around the world. And unfortunately, approximately every 45 seconds in this country, someone succumbs to heart disease. So this is obviously a major health problem in our society. And what we would like to be able to do is to, using various methods, such as behavior modification and certainly medical intervention, bring down these numbers so it's no longer as prevalent a problem as it is at the moment. 

The good news is that over all, deaths from heart disease have decreased. These particular numbers are from 1993 to 2003. It was found that cardiovascular disease death rates dropped about 22%. Anybody suggest why? Statins, yes. But it's smoking actually, convincing people not to smoke has made this much of a difference in cardiovascular disease rates in this country. So what are the causes of heart disease? What are the risk factors? Well, there are things we can do nothing about. The things that we cannot change are the genetic factors, the genes that we inherit from our parents. 

I'm come from a family with a history of cardiovascular disease. My father's mother died of a heart attack. My Aunt had a heart attack, and fortunately didn't die from it. My dad died from a ruptured aneurysm. So all of these things, we cannot escape. And of course, with increasing age, there's increasing risk. And I apologize, gentlemen, but we're at much greater risk than women are, particularly in advanced age, for having heart disease. But there are things that we can change that will reduce our risk of having heart disease. We can avoid high cholesterol. These are the statins that were mentioned. 

These are very prevalent now in our society. Half of my family is on Lipitor at the moment. Blood pressure, something I also inherited from my dad. That can be treated in a couple of ways. The best way to treat it is exercise. But if that doesn't work, you can also take medications that will reduce your blood pressure by reducing your blood volume. That is, diuresis and diuretics. Diabetes, unfortunately if you have it, it will increase your risk of heart disease. Smoking, definitely do not. And diet, a high fat diet, and certainly in people who are presently obese, this is a significant strain on the heart. 

Carrying around the extra weight leads to cardiac hypertrophy and ultimately heart disease down the road. Don't be a couch potato, because if you get out there and exercise, even just walking around. This is a good reason to have a dog. If the dog has to go out and walk around, then so do you. It's a very good idea. Being a dog owner myself, I can assure you that that's a good thing. A little bit of alcohol is a good thing. A lot of alcohol is a bad thing. So it's on the order of one to two drinks per day, will actually reduce your risk of heart disease. But if you drink a lot more than that, that can be a real problem. 

And then something else that we face every day in our society is stress. And stress will increase your risk of getting heart disease. So you can medicate, exercise, many different things help to relieve stress. So these are the things that we can change. And these the things that we cannot change. And throughout my talk, you'll notice references to Web sites that are very useful. The American Heart Association has been a very strong proponent, getting out to the public, raising funds, making people aware. Their big drive now is to make women aware of their risks for heart disease. So when we talk about heart disease, what are we actually talking about? Well, most of us, when we think of heart disease, think of coronary heart disease. 

And this is something that most of us are aware of. It's fairly routinely on television. It's available to you on the Internet. Information about atherosclerosis, the presence of fatty deposits in your coronary artery walls that ultimately build up and cause an occlusion or a blockage, which leads to the cutting off of blood flow from a certain area of the heart. This is what I refer to as acute heart disease, because it builds up silently within you and then all of a sudden, you have this major chest pain, or you don't feel quite right. And that's the onset of a heart attack. Other types of heart disease I put into a chronic category. These are long-term problems. The most common of these is a cardiomyopathy called Dilated Cardiomyopathy. So the function, the heart, in order to function well and move blood around the body, has to have a very strong wall, which contracts regularly from about two weeks of gestation up through your very last day. 

And it normally has ventricles that are a certain size. And the combination of the muscle and the size of the ventricles makes it a very efficient organ for pumping blood. As you can guess from the adjective "dilated," that what has happened here is the ventricles have expanded significantly. And the other problem is that the walls of the ventricles have thinned significantly, which makes the heart much less efficient in terms of its necessary function. That then, leads to lots of other problems. Also within this category, we can put ischemic heart disease. So ischemia refers to an area of the heart that has been cut off from blood. And basically, that is becoming connective tissue, rather than healthy functioning muscle. And congestive heart failure refers to a particular long-term problem which leads to a build up of fluid in the body. It can be controlled, surprisingly enough, with medication. 

These are the things that we think about when we think about heart disease or cardiovascular disease. But this is just a very brief list. If you want more information, go to the Internet. Look up WebMD. Go to the American Heart Association Web site. There are actually a very large number of very good, well informed Web sites that will give you all of the information that you'd be interested in. So this is one example of cardiomyopathy. At the top, we have a normal looking heart with the ventricles and atria opened up, and also the aorta and pulmonary artery. And you can see that the walls are nice and thick and robust. And there is a relatively, look at the volume here of the ventricles. 

Compare that down to the bottom, where we have enlarged ventricles. Notice the thinning walls. This is part of the problem. Now if it works, I'm going to show you a little cartoon of atherosclerosis, and how it builds up over time, leading to what we call a heart attack or MI, a myocardial infarction. I'm not getting volume here. I'm wondering if there is a connection, because you guys can't hear this at all, right? It's part of it. Well, what I can do is do it through my microphone. Just turn this up. I apologize for the delay. 

- Atherosclerosis is a contributing cause of coronary heart disease. It is characterized by fatty lesions, which grow within the walls if the incised arteries, a class which includes the coronary arteries. These lesions, or plaques, form in a layer of the arterial wall called the intima. The intima consists of endothelium and a thin layer of active tissue, or collagen. The high turbulence of blood at the bifurcations of the coronary arteries begins the process of atherosclerosis by entering the endothelial layer. Injured endothelials increases its permeability to monocytes, which migrate into the intima and change it to macrophages. These macrophages express receptors for oxidized low-density lipoproteins, or LDLs. Injured endothelium also increases its permeability to LDLs, which in the presence of free radicals are oxidized and bind to the macrophages. The macrophages continue to absorb oxidized LDLs. And enlarged, become foam cells. 

These macrophages also secrete substances which cause the migration of smooth muscle cells from the media and the proliferation of smooth muscle and collagen in the intima. The presence of foam cells and smooth muscle cells gives the appearance of a fatty streak. The further proliferation of smooth muscle cells and collagen, and an increased absorption of lipid leads to formation of a fibrous plaque. As the fibrous collagen layer expands, necrosis occurs as the cells inside the lesion die from lack of oxygen. This lipid-rich plaque, the necrotic center, is called an atheromata. Eventually, the necrotic region is walled off by calcification of the plaque base, and a thickening of the fibrous cap, giving rise to the complicated lesion. A rupture of a lesion induces clotting, which begins with the aggregation of platelets, followed by the accumulation of fibrin, forming a thrombus. If this thrombus blocks the blood flow, a coronary infarct will occur. 
- I apologize for the not-so-wonderful quality of the images. We had to turn down the resolution of the computer earlier on, and that's why they weren't quite so nice. 

But I think you got the general idea of what we're trying to get at. Over time, because of a high-fat diet and too much cholesterol in the blood, and normal physiological arrangement of how the blood flows through the coronary arteries, you get an abrasion to the blood vessel wall, which leads to invasion of the cells called macrophages, which are beneficial cells normally. But under this circumstance, because they reside, and they start to take up LDL, what happens is you have a build-up of a fatty deposit in the wall, which gradually gets larger, ultimately leading to a situation where it will rupture, causing a clot to form. And that clot is what causes the problem, which is the occlusion of the coronary vessel, cutting off a small or a major portion of the heart wall itself, which without direct, immediate intervention, ultimately will lead just to scar tissue taking the place of normal, healthy muscle. And of course, that significantly reduces the ability of the heart to contract and to function. So what is the present and for some fairly longstanding treatment for this problem? You've probably heard of bypass surgery. My cousin's husband just had what is normally referred to as a massive heart attack. He was out playing tennis with his kids. And fortunately one of his kids is an ER nurse. 

So when he had this heart attack, she immediately got him to the ER and they had to do a triple bypass. Thanks to early intervention, he's doing extremely well. And probably lost a lot less of his heart muscle than he would have otherwise. So bypass surgery is common. Stent placement is used to open up the occluded vessel and keep it open. So it's sort of like a spring that you put inside of the vessel. Then, when it opens up, it actually is a support to keep the vessel open, so that blood can continue to flow. Another possibility is transplant. But there are too few hearts and too many people who need them, so this has not really been a good option in most circumstances. So often what happens, is people who have heart disease go on some kind of pharmacological intervention. 

One of the obvious ones is keeping your blood thin. So doctors will actually prescribe for people who are risk to take an aspirin a day. Aspirin thins your blood. This reduces the possibility of clotting. A glass of red wine will help thin your blood. So if you need an excuse to have a nice red wine with dinner, here it is. Of course, anti-hypertensives, you want to keep your blood pressure down because the higher the blood pressure, the more at risk you are for certain problems. Statins, everybody's favorite. And also drugs that will regulate or control heart contraction and of course chest pain. So that's what's going on now. 

What's going on behind the scenes? There are a number of clinical trials for drugs and other therapies. If you're interested, you should go to: clinicaltrials.gov They list everything nationally and internationally. And basically, using that list, which is quite lengthy, what I'm presenting to you here is the idea of using your own stem cells for your own treatment. So what's happening here is that most frequently, stem cells are being taken out of your bone marrow. So that's a relatively common procedure nowadays, where an MD will simply go in with a needle into your hip and aspirate some of your bone marrow, which is full of cells. Not all of those cells are stem cells. But the stem cells can be isolated, and then those stem cells can be used to treat a variety of diseases. I'm listing this one about the endothelial progenitor cells for chronic heart disease because this one is going on in Madison. So just in our back yard, Dr. 

Lavalle is heading a study in which the cells that will make the cells that line your blood vessels, that are called endothelial cells, he takes the precursors, or progenitor cells to those, out of the blood. He then uses those and injects them into the heart of individuals that have a specific type of chronic heart disease. And what the idea behind this clinical trial is that the blood vessel cells will restore blood vessels to the ischemic heart. Thereby increasing function and prolonging the life of the patient, making the patient's life better. So that's a particular type of stem cell. Yeah, go ahead. 
- The vessel and wall, nothing to do with the heart muscle? 
- Not to do with the heart muscle. But you cannot have heart muscle without a very extensive blood supply. So each cardiac muscle cell is associated with four different capillaries. 

That shows you how much dependent the heart muscle is upon its blood vessels. So improving the blood supply to the heart will improve the function of the heart, not directly to the cardiac muscle, but overall by affecting the overall supply of nutrients and oxygen to the heart. And the other major type of study that you will find on this particular Web site is for bone marrow or mesenchymal stem cells. So mesenchymal stem cells is a very general term. It doesn't refer to a very specific cell type. It refers to a broad group of cell types that can be defined by the proteins that they express on their surface. And frequently, these mesenchymal stem cells come from the bone marrow. They will come out of the bone marrow and into the circulation. Another source that has been discovered within the last few years for these mesenchymal stem cells is fat. And you say, great, everybody has fat. 

So all you've got to do is go into those areas. There are two types of fat. One is white fat, which is most of what we have. Then there's brown fat, which we have a lot more of when we're a baby. But as an adult, you still have some, like underneath your shoulder blades, there's brown fat. So it's been discovered that within these areas of brown fat, and perhaps even in the white fat, you have stem cells. And these stem cells are your own. So they can be used. All they have to do is be isolated, and perhaps stimulated in some fashion. They could be used to make more cardiac muscle, or more blood vessels. 

And I noted that one planned study on this Web site actually suggests that they will isolate cardiac stem cells from the heart directly. And I will tell you about these stem cells in just a minute. Or to expand in a tissue culture dish and then inject them back into the patient. That's not yet started. So those are the clinicals. I've shown you the traditional methodologies that doctors are using to treat heart disease. I have now talked to you about ones that are in planning, are in real planning, in clinical trials. And now I'm going to talk to you about things that will happen in the future at some point, but probably not for five or ten, or maybe even 15 years. I think we've all heard about human embryonic stem cells. They're quite controversial. 

They have been a focus on this campus, thanks to the work of Jamie Thomson and his collaborators. Jamie was the first to isolate and culture human embryonic stem cells. And the beauty of these cells is that they can give rise to any cell type you want, from neurons, to bone, to cartilage, to heart, to skeletal muscle, anything you want, liver, kidney. These human embryonic stem cells can make these cells. And they can make endothelial progenitors, cardiac stem cells, neuro stem cells, etc. But there are some issues. I'm not going to go into detail about that. But they're not easy. And they probably will not, in the near future, be for any sort of clinical therapy. And nobody is proposing to do that. 

But they are a possible source, because they are an unlimited source. You can make as many of these cells as you want. And that is the beauty of it. But more recently, Dr. Thomson and several other labs, a lab in Japan, and a couple of other labs in the US have reported the isolation of what is called induced pluripotent stem cells. What does this mean? It means that I could take a biopsy of your skin and put it into a tissue culture dish, and those cells would grow. And then I would genetically modify those cells with three or four different gene products, and be able to isolate from those modified cells, things that look almost exactly like embryonic stem cells. Except they're yours. They're nobody else's. 

So the potential here is that these induced pluripotent stem cells, because they are patient specific, and because they don't require the destruction of any embryos, they're not controversial. They're not unethical. And they are patient specific, so you don't have to worry about immune responses. Potentially, these may be the cells that will be used in cell therapies of the future. But again, we have a lot to learn about these cells. They've just recently been isolated. And probably will not be used for at least ten years down the road, unless something unusual happens. But it's always good to know what's happening in the future, or could happen in the future, so that we can focus our energies in a way that would be most productive, particularly in the research field. So, I've been talking a lot about stem cells. What are stem cells? 

We hear stem cells almost every day in the news, it seems. On television, magazines, on the Internet. What is a stem cell? A stem cell is quite an amazing cell in that it can replicate itself and make identical copies of itself or, under the appropriate stimulus, give rise to cells that will then go on and differentiate to form neurons, to form blood, to form muscle, etc. So stem cells are called pluripotent, because they have a very wide range of cell types that they can form. This is an incredible source of cells and tissue that could be used in a clinical setting. So, just a few definitions to help clarify what we're talking about. Stem cells are self-renewing. That means they can make copies of themselves for years on end, with no problem, without differentiating. They are pluripotent or multipotent, depending upon how many different cell types they can form. 

So pluripotent cells from an embryo are thought to be able to form any cell type in your body at all, whereas multipotent stem cells might be able to form, say, endothelial cells, and blood cells, and cardiac muscle cells, but only those three types. That's the difference between pluripotent and multipotent. Then once a cell has taken a step away from a stem cell, it is generally thought of as a progenitor cell rather than a stem cell. It has limited ability to self-renew. It will only self-renew so many times before it no longer can. And it's usually multipotent. That is, it can only make two, three or maybe four different cell types. Then we have yet another beast, which comes from stem cell to progenitor. Then we have the transit amplifying cell, which come from these two. And these have a very limited number of times they divide before they actually differentiate. 

So if you think of your skin. You have stem cells on the very bottom layer. Then when they divide, they have to give rise to all of those layers of skin cells that protect us from the outside world. And you can think of those stem cells, at least at the bottom, as being transit amplifying cells, which then lead to the differentiated cells that we recognized as the skin on the outside of our body. And differentiated cells are unipotent cells. What's kind of amazing is there are at least 200 different types of these cells in our bodies. So what's a cardiac stem cell? Well, a number of years ago, a gentleman rediscovered for the world that the heart can divide, the cells in the heart can divide, but to a limited extent, which was a shock, because the Dogma that even I thought ten years ago, said that once you were born, you couldn't get any more cardiac muscle cells. He said, no, that's wrong. With normal wear and tear, the heart will replace cells that get worn out. 

But if you have heart disease or have a heart attack, there aren't enough stem cells there to replace what is lost. It's only there to replace the occasional day-to-day cells that are lost in normal body functions. So cardiac stem cells are multipotent. You want them to be able to form three different cell parts. Most important, the cardiac muscle cell, but also endothelial cells, because you need a blood supply. And smooth muscle, because you have very important coronary arteries, as we just learned about in the cartoon not too long ago that supply the blood to the cardiac muscle. So a cardiac stem cell would be considered multipotent, because it can make these three different types. Another definition of a stem cell is that it needs to be self-renewing for a lifetime supply. So we have to have these cells around for our entire lives so that we can have replacement of myocytes as needed. In a scientist's definition, you have to be able to take these stem cells out of the body and put them in a tissue culture dish and grow them, while maintaining them as stem cells. 

Because ultimately, you want to expand the numbers so that you can do a cell therapy with these cells. So they need to be expandable. And finally, they need the ability to functionally repair the region of the heart attack, the ischemic area of a myocardial infarction. That is where my daily research life revolves around. And there are hundreds of other labs around the world that are also putting their best minds together to try to figure out how this will work. So, where do these stem cells come from? First, I'm going to tell you about the ones that actually live in the heart, which you say of course is the most logical place for them to be. So, frequently we'll use rodents in the lab as a model to look at things like the cardiovascular system, understand its development and differentiation and function. So the focus has been on the mouse or the rat. And basically, what I'd like you to take home here is not the names, which are really not important. 

But understand that there is a developmental sequence. There are cells that live in and among the muscle cells in the ventricle and the atrium. And they express certain proteins on their surface that mark them as stem cells, that says it is a stem cell. That is the only thing at the moment that says this is a cardiac stem cell. It does not make any cytoplasm proteins. And it does not make any nucleic proteins that say this is a cardiac cell. All it says is this is a stem cell. Now, when this cell stops being a stem cell and divides, and starts the process of differentiation, not only does it maintain the stem cell antigens or proteins on the surface, but it now starts to express the nucleic proteins that say cardiac or smooth muscle. Then the next step in the process is the expression of cytoplasmic and membrane proteins, but they continue to express the stem cell markers as well. Then the final step kind of becomes a differentiated definitive muscle cell. 

It loses the cell surface markers that say stem cell. It only makes the cytoplasmic and the nucleic protein. So this is a process. It's a way to think about things. You have cells in the heart that are sort of sitting there, waiting for some kind of a signal. They will divide occasionally, but not often. But when they get a signal, they go from a stem cell, to a progenitor cell, to a precursor cell, and ultimately to a differentiated muscle cell. This is how the process works, and how it has been characterized so far. And all of this occurs in the mouse, but it also occurs in us. It's just a lot harder to study in humans. 

What do these cells look like? This is always cool when you can see what it actually looks like. On the outside, you have muscle cells, which are very large cells. They're stained red because they're stained with what's called an antibody. And the antibody recognizes a particular protein. Then it's labeled with a fluorescent marker, which could be red, green, yellow or blue. And what is unique about the stem cells is they're very, very small. So most of the cell that we call a stem cell is a nucleus. It has a very thin rim of cytoplasm around it. That's true of all stem cells. 

They're mostly nucleus, and very, very little cytoplasm. This was stained for a couple of different proteins. These are two nucleic proteins that are cardiac muscle specific. This is a stem cell cartoon. Don't worry too much about the colors and where they are. But I guess what I'd like you to take home is that in the heart, there are little pockets of these stem cells. And they are sitting there, occasionally dividing, but they're waiting for a signal, which says divide, amplify and replace some cells over here which are about to die. This is normal. This happens throughout your life. 
(inaudible)

 
- In the individual stem cell, progresses and differentiates, it's gone? In other words, there's a limited supply? 
- There can be a limited supply. That's actually a very good question. So there's a group of stem cells. Let's say there are 1,000 stem cells. And over time, they will start to diminish. But they're very robust, particularly when you're growing, because your heart has to increase in size. But then, once you get to be an adult, you're not going to grow that much any more. 

Then they would sort of plateau. And by the time you're say, 60 or older, they're going to be in decline, but they're still going to be there in some numbers because they divide and replicate themselves at the same time that they divide and make cells that will go on and make muscle. Very good question. Yeah? 
- Stimulate that continuing debate. 
- You got it. Excellent. Would you like to come and work in my lab? 
(laughter)
 Let me tell you a little bit more about-- I'm sorry, what? 

(inaudible)
 
(laughter)
 Okay, so what is this place that the stem cells live look like? Now I'm going to give you another vocabulary term, which you've probably heard in a different context. This is the stem cell niche. Everybody in the stem cell world thinks about stem cells in the context of a stem cell niche, which means sort of a home and a supporting structure of extracellular matrix, and also more importantly, other cells. So here we have the heart. And basically, we're looking at an area here, which is now in an expanded view. These red cells are the cardiac muscle cells. 

The cells right here in the middle that have what's called a basal lamina, which is an extracellular matrix surrounding them, those are the stem cells. But they don't want to live all by themselves. They want to be surrounded by cells that they know and love. So you have these other cells. And you also have these amplifying cells. So these cells that are larger and kind of purplish in the middle are no longer stem cells. They've taken a step away from the niche. They walked out the door and they can't go back. The stem cells that are here stay, and continue to make more of themselves, whereas these guys will go on and make cardiac muscle cells, endothelial cells, or smooth muscle cells. So what do these cells need? 

They need to be close to each other. There are proteins that connect cells. And one of them in particular seems to be very important in maintaining stem cells as stem cells. But you also have to have things like gap junctions, where the cells are directly in cytoplasmic contact with each other. And you have to have other things. You have to have other cell types, maybe cardiac fibroblasts. It's not entirely understood at this point. But what is emerging is kind of a support structure, if you will, for the stem cells, which we call the niche. What do these cells look like? Can you can actually see them in the heart, rather than just in a drawing. 

Yes, you can. This is a paper from 2005, published in Nature. And basically, they were staining for a particular protein called Violet 1. And if you look in here and you see the dark brown nuclei, the dark brown stained cells, those are the stem cells based upon a nucleic protein that they're making. So we can actually see these things. We're starting to understand what they look like. So now the task of the scientist is to recreate that stem cell niche outside the heart in order to be able to expand the stem cells, from a particular patient, expand them and then put them back into the heart of that patient to see if that will benefit the patient by regenerating cardiac muscle and blood vessels. So all around the world, are popping up study centers for regenerative medicine. This is the new wave of medicine, regeneration, figuring out ways to get organs to make more, to regenerate their cells, that are part of their normal functioning day-to-day life. So what has been done is you take everything but the cardiac myocytes from a heart, from a rodent, and you put that in a tissue culture dish. 

Then you take out the stem cells, based upon the proteins that they express on the surface. You put them into culture, and you follow them to see if there are ways of detecting if they remain stem cells. And of course, if they go off and differentiate into something, you can see spontaneously contracting cardiac muscle cells. And there are also ways of detecting smooth muscle for the vascular and endothelial cells, as well. What has been found, as we look at these things, is that they won't just grow in any old tissue culture dish. They need special media conditions, in particular growth factors, which we sometimes call cytokines, are important for culturing the cells and maintaining them as undifferentiated cells so that they can be expanded. This is a diagramatic way of thinking about it, which often helps, because words sometimes generate different pictures in different people's minds. So if you start with what we call a cardiac progenitor cell or cardiac stem cell, it expresses certain proteins, one on the surface and one on the nucleus. It will go on and some will stay stem cells. That's what this little diagram means. 

The arrow out to the side is the duplication of the stem cell. But another stem cell will divide and go on and make what we call mesoderm, which is what makes all of muscle. And then, within that mesoderm, you have a differentiation into a lineage that will make a smooth muscle, cardiac muscle cell, and an endothelial cell. This is the cardiac lineage. This is the smooth muscle lineage. This is the endothelial lineage. So this is a conceptual way of thinking about stem cells, progenitor cells, to transit amplifying cells, to differentiated cell, if you will. In the meantime, of course, these cells can be used for drug studies and other things that will ultimately benefit people down the road for different therapies. Because if we could give a drug to a person which would expand their stem cells, that would be a huge benefit. It would allow them to do things, like regenerate parts of their cardiac wall. 

That's the ultimate goal. You want to do this in a minimally invasive fashion. So if you need to put in cells, that's one thing. But if you can simply give a drug that will stimulate the cells that are already present, that's even better. And if you can do something that's not a drug and have side effects, that would be even better. That's the sort of way we're thinking about it. Now, because we don't know precisely what a stem cell looks like, a cardiac stem cell, we don't know all the defining features. We can't isolate a cell and say yes, this is a cardiac stem cell, without killing it. Because to define it, you have to get inside to the proteins that define the cell, as far as we know it. So you don't want to do that, because you want to keep the cell alive. 

What other labs have done is to use a transgenic approach in rodents to define these cells. And yes, they do exist. They exist based on a definition of what genes and proteins they express. And the bottom line here is that these cells can form all three types, the cardiac myocyte, the endothelial cell, and the smooth muscle cell. Two different labs have found this using completely different approaches. Another lab found that you could get at least smooth muscle and cardiac myocytes. The way that development works is you get a gradual narrowing of the capability of the cell from the pluripotent cell to the adult cell. So here, they have found a cell that is multipotent and can make three cell types. Both of these two labs did it independently. This lab said it could make at least two cell types. 

So those are the adult sources. To summarize, the cells that live in the heart is what we have just discussed. And now what I'm going to talk about are the so-called mesenchymal stem cells, which come from bone marrow predominantly. But they can come from other sources in the body, such as fat. What's in bone marrow? All kinds of wonderful stuff. Most of us think of it as a source of new blood cells. Perhaps where red blood cell precursors come from, as well as white blood cell precursors. And cells that will make blood vessels. And there are these other things that have been defined by a group in Minnesota, called multipotent adult progenitor cells. 

It sounds very impressive. They don't make heart, so they're not that impressive, at least in my book. But they do make lots of other cell types. And then we have this sort of vaguely defined mesenchymal stem cell population. I'm sure there are other cell types which have not yet been discovered which like to live in the bone marrow, as well. So don't worry about these names. But what was interesting was this particular group did a study. And what they got to occur, was that they mobilized the cells that normally are happy and comfy in the bone marrow to get out of the bone marrow, come up to the heart because the heart has problems. They damaged the heart and then they used certain cytokines or growth factors and got cells to come out of the bone marrow, hone in on the heart, and contribute to the cells within the heart. Isn't that amazing? 

(inaudible)
 Yes, just on rodents. But you know people are thinking about how to do this in people. We just don't know exactly what signal to send. So a question that I posed was you have these cells which expressed the same thing on the surface in the bone marrow and in the heart itself, what's the difference between them, or are they the same. Is there really an endogenous population, or does it all come from the bone marrow? Or does it only get supplemented by the bone marrow? It's an interesting question. And there are other cells that exist in the bone marrow that we have not yet identified in any way, which nonetheless can contribute to cells in the heart. So what is it that stimulates these cells to take part in cardiac regeneration? 

There are these growth factors, cytokines, called stem cell factor, or granulocyte colony stimulating factor, G-CSF for short. And again, if you give a mouse or a rat a myocardial infarction, usually the way that's done is by tying off one of their coronary arteries, which basically mimics what happens in a human heart attack patient, and then give them one or both of these growth factors, they found that rats did better. And the way that the group explained it was there are things coming out of the bone marrow that are playing a beneficial effect directly with cells of the heart. Mesenchymal cells are usually bone marrow derived. They circulate in our blood. They have been found in umbilical cord blood. There are a number of people who are starting to save umbilical cord blood from their kids, with the idea that down the road, maybe the kid's going to need some stem cells, and this is their only stem cells, right here in their own blood. It's an interesting thought. These cells, once you take them out of the body, are very happy to grow and expand in numbers. But they require something in what we call the micro environment that will stimulate them to do what we want, because they can do lots of things apparently. 

They can make bone. They can make fat. They can make cartilage. But if you want it to make heart, you have to give them a certain combination of things, either cell-cell contact or what we call paracrine factors, which are locally secreted growth factors for which there are receptors on these particular cells. The wonderful thing about mesenchymal stem cells is that they suppress the immune system. They prevent an immune response. So what has been happening in certain clinical situations, for people who have, for example, a kidney transplant, at the same time they will get an injection of mesenchymal stem cells from the bone. And the presence of those stem cells at the same time as the transplant actually suppresses the immune system and leads to tolerance of the transplanted organ. This is how it does it. I'm not going to go into this in detail. 

But generally there are several mechanisms by which mesenchymal stem cells can suppress the immune response, and thereby facilitate either a cellular or organ transplant, which is really cool. Brown fat, or adipose tissue. So the stem cells are thought to be much higher in concentration in brown fat than in white fat, but they do exist in white fat. Brown fat is much less in us than when we were kids, but we still have some. They're multipotent. They can form cardiac myocytes, vascular smooth muscle and endothelium, which is exactly what we need. And if you were to look at a mouse model, which is the results are shown here, they were basically looking for three different cell types that arose from the adipose stem cell. Basically, this lab isolated adipose stem cells based on a surface marker and injected them into a rat. And I don't recall if it was directly into the heart, or if it was just into the peripheral circulation. But what they found was that these cells were able to graft and do all of the things that needed to be done, such as making gap junctions, forming myocytes. 

And all of these are antibody stained. I won't go into it in detail. But this is the sort of evidence that is presented when you say, okay, this is a stem cell. I put it into a mouse model, there you are, you've got cardiac muscle, and all of the different cell types that you need. So we know it works. And the question is, do we really need the cells at all, which alludes to something that I mentioned earlier. An interesting observation over the years has been that no matter what type of cell type you put into an infarcted heart, it helps. It reduces the amount of scarring and overall it helps the function of the heart. Why in the heck is that? You would think you'd want to put in something that you would normally find in a heart. 

You don't have to. You can put in skeletal muscle cells. You can put in bone marrow cells. There are a number of other cell types that have been tried. And all of these have led to some benefit, which says it can't be the cells by themselves because there are too many different cell types. Now we're asking what it is that the cells secrete when they're implanted that makes the difference. Because you don't actually get many cardiac muscle cells actually forming, but you do get a much greater benefit to the functioning of the heart than you would expect, based upon the number of cells that differentiate. And not only do they replace the cells, they also seem to prevent other cells from dying. They're secreting something which says to the cardiac myocytes that are in there, you don't have to die. This is what we call a paracrine signal. 

This is just a little diagram. Many of us have heard of endocrine, which is at the top here, which is say, your thyroid gland is making thyroid hormone, which then goes down quite a distance in the blood and acts on cells at a distance. Paracrine signaling is at the upper right here. Basically, a cell makes a growth factor or cytokine and then just one or two cell distances away from that, there's another cell that has a receptor. This is what we mean by paracrine signaling. It's short distance signaling that makes a difference when you're transplanting cells into the heart. I told you about the adult cardiac progenitor cells. I told you about the bone marrow mesenchymal stem cells, also some brown fat stem cells. Let's now talk about embryonic stem cells, the controversial ones. Embryonic stem cells are derived from embryos. 

They are the part of the embryo that will give rise to everything in the body of the organism itself, not the extra-embryonic, not the placenta, not the amnion, not the yolk sac, none of that, just the cells in the embryo proper. And it's been found that these can be cultured outside the animal, with no loss of pluripotency for years. No other cell types do this, except cancer, and that's a completely different mechanism. Cancer is not normal. These are normal cells. They can differentiate in the tissue culture dish to form cardiac progenitor cells, and cardiac myocytes. And myocytes that are in these cells as they differentiate are in clusters of cells. So it's not like you get an entire patch of muscle cells all together. They're intermixed, which actually makes them a little bit difficult to manipulate, because you have to purify the muscle cells away from the rest. This is a way of thinking about it. 

You start with, say, a human embryonic stem cell, which is pluripotent. It expresses certain proteins that say it's an embryonic stem cell. It goes on and makes more of itself, but also sometimes, it divides and starts to differentiate. It will form what we call either endoderm, mesoderm or ectoderm. The muscle comes from the mesoderm. So it differentiates to form a mesodermal stem cell, which expresses a certain nucleic protein. Then it goes a step further and becomes more limited to blood and heart. Then it expresses something else. And some of these will go on to form exclusively blood, or blood and endothelial cells. Another branch will go and form smooth muscle cells. 

And some of these will go on and form cardiac progenitor cells and ultimately cardiac myocytes over the course of about a week to ten days in tissue culture. So that is one view. This is a similar view, but slightly more colorful. You start with an embryonic stem cell and then you gradually progress through the accumulation of different membrane proteins, cytoplasmic proteins, and nucleic proteins. I don't want you to worry about the names, just the overall process. Some of these will then become mesodermal cells. And then ultimately, our hope is that we can find the right combination of membrane protein markers on these cells so that we can say, this is a cardiac muscle stem cell. We haven't found that yet. Ultimately, that will form the cardiac muscle cell. It might come from more than one direction. 

It may not come in a linear fashion. It may have some flexibility in the way that these cells develop. This is not a great picture. It's something in a fluorescence staining of different cell proteins within cardiac muscle. It shows you that indeed you can get cardiac muscle from the cells. And one of the things that various groups have shown is that you take different types of stem cells and you inject them into a mouse model of myocardial infarction or heart attack, that you can get these stem cells to form the cell types that are needed to regenerate the heart. So, whether it's undifferentiated stem cells, or mesodermal stem cells, or even cardiac myocytes that have differentiated, all of these can be injected into an area of myocardial infarction and they will sit there and become functional parts of the heart. A number of labs, including our own, have shown that in a rodent model, this is the way it works. And it can work. Therefore, in principle, it should work equally well in people. 

I'm not going to go into this. This is simply marks the cells to show that they have stayed where we injected them, and that they have differentiated. This is the end result. The most important thing is function. It's nice if you have different cell types in the heart. The most important thing is improved function. And what we're doing here is we're looking at the diameter of the heart after contraction. And we're showing that injection of cells improves, that is, makes smaller the diameter, so that the blood flows out of the heart more efficiently. Then this is a measurement of the shortening of the muscle. Again, after heart attack, injection of cells increases the rapidity of shortening. 

It puts them back into a normal range of function, which is shown here at the left, gradually getting improvement of the physiological parameters of the heart. That is the bottom line. That is what is most important. And what we showed was that mouse ES cells in the mouse heart can survive, differentiate and form all three types of cells that we needed. More importantly, it blunted remodeling, prevented scar tissue from forming. And we showed in a separate paper that it increased survival of the cells that were already there. We were able to do this with a relatively small number of cells. If you're going to be doing an invasive therapy that's introducing cells, you want to minimize the cells that you have to inject, simply because you don't want other funny things going on. The less cells, the less likely there is to be a problem. You have to have enough cells to get a response that is desired. 

The problem with ES cells is tumor formation. We don't yet have a way of producing, selectively producing the cells that we want. If they are used, because they're not from the individual, we will need to have immunosuppression. And one of the ways that we can think about this is to increase the number of ES cells that we have, that will match all of the major histocompatibility types that represent an immune system and a response to anything that is introduced into the body. These are called the HLA markers in people. We need to think about tolerance. That is, making the immune system accept these cells, and not reject them. So you can see the use of human embryonic stem cells, as we have them now, faces a lot of hurdles, aside from the ethical ones, before anything happens with them in a clinical setting. Yeah? 
(inaudible)

 
- Are the tumors cancerous? No. 
(inaudible)
 That's something different. In this particular explanation, embryonic stem cells, because they are pluripotent, make lots of different cell types, a lot of different cell types. They make neurons, and bone, and lining of the gut, and everything else. And sometimes, these cells will make little tumors. They're not what we think of as tumors. They're benign structures that have lots of differentiated cell types. 

The only threat that is posed by them is that they will interfere with the function of the heart. You don't want to have a blob of cells growing on the outside of your heart, which is potentially what could happen here. But yes, if I can get back to your question about stimulating cells into a cancer, that's a completely different subject. 
(inaudible)
 Perhaps, as you wish. Since we can't use ES cells, a new player on the scene is the induced pluripotent stem cell. These were just recently isolated in a number of labs. There was the big broo-ha-ha, even on the regular news saying, wow, look at this. And what are these cells? These are just cells that are growing in your skin, what we call the dermis part, underneath the epidermis. 

You can take these cells and transform them into pluripotent stem cells by introducing a couple of different gene products. The Thomson lab used several nucleic proteins. Other labs have also used nucleic proteins, but different ones. So there's some combination which will work, and it's not just one combination, it could be several different things. The problem is that when you're reprogramming these cells, which is what you're really doing, you're changing their phenotypes so they're no longer a fibroblast. They're not a skin cell anymore, they're a stem cell now. And that stem cell has the same abilities to make cardiac, smooth muscle, skeletal muscle, neuron, glial, fat, bone, etc. But they have been genetically manipulated. Could this activate something else? Yes, possibly. 

That's a major concern. So we'd like to be able to do this, but we'd like to be able to do it in a different way. People haven't yet discovered how to do that in a way that does not require genetic manipulation of the cells. It's something that's a very intensive subject of research. But what could it do for us? Even in the beginning, we can start to analyze skin samples from people who have diseases. And it will allow us to have a population of cells that will recreate, in a tissue culture dish, what goes on in the diseased individual, and thereby will allow us to access new knowledge to the process of disease and its development in some patients that we don't know very much at all about how the disease works. Down the road, because these cells are patient specific, they will not induce an immune response. You can take these cells and put them right back into the patient for their benefit. And they won't suffer any adverse effects due to the immune response, which is really exciting. 

But again, this work is down the road a ways. Let me finish up, because I'm probably running over. I like to talk. So you can get cardiac stem cells from several different sources. There are some that live in the heart. There are some that live apparently in the bone marrow and in other places such as adipose tissue. And outside sources, or different sources, are embryonic stem cells and induced pluripotent stem cells, the wave of the future. All of these appear to be able to differentiate into three different types. And we have found that they have different requirements that we're still learning about. And as I've said several times during the course of my talk, a definitive marker for cardiac stem cells has not yet been discovered. 

They express stem cell proteins, but we haven't yet found the combination that says it's a cardiac stem cell versus liver stem cell, for example. That's the end of my talk. I'll be happy to answer any questions that I can. Remember, I'm not an MD. Yes? 
(inaudible)
 
- The study that's going on here. 
- Oh, yes, the clinical study on the endothelial progenitors. 
- You get the stimulation of blood vessels? 

- So what happens is the doctors take blood from the patient, and isolate from the blood, certain cells that have certain surface markers. These are called endothelial progenitor cells. When they differentiate, they go to form endothelial cells, which line our blood vessels. They can make new blood vessels. 
- If there's already an area of infarct and compromised muscle, aren't those cardiac cells already dead? 
- Yes. 
- So then how does increasing blood flow to a dead area help anything? 
- A good question. What I would propose to you, I can't give you a detailed answer about that. But what I would propose to you is if you can induce an area that has poor blood supply to form new blood vessels, that will permit the opportunity for other stem cells to get in and repopulate the area. 

That's a simplistic way of looking at it. But I think that's what the goal is. 
- The second question was in relation to the issue of differentiation and the potential for things going off in the wrong direction from the stem cells. Does the area of epigenetics intersect with these issues? 
- That's a very complicated question. Under normal circumstances, as stem cells differentiate, if they do something they're not supposed to do, such as form a cell that is inappropriate for that particular location, there are cellular mechanisms that will lead to what is called cell death or apoptosis. The body has its own internal mechanisms which prevent cells from doing inappropriate things. So sometimes when cells divide, not all of the chromosomes go into each cell, for example. And those sorts of cells, because of what we call cell checkpoints, would end up dying and being re-absorbed by the surrounding tissue. That's the normal situation. 

Now, you go outside, you breathe in diesel exhaust and the stuff that's coming out of factories, and you drink water that has mercury introduced into it through the burning of coal, and it's all rained down into our ponds, and you have the loss of certain chemicals into our environment, that's where you have problems. That's where you start to have things happening. And I can't say much about this, because I am in no way an expert. There are experts on campus. And if you want to hear people talking about stem cells in cancer, I think you should invite people along that line. There are several different people on campus who are very much interested in cancer stem cells, which I could easily try to talk about for a much longer time, but I'd be wasting your time because I'm not an expert. But yes, these things can happen. But normally, they should not happen. And it's only when something outside the body, for whatever reason, gets in and influences the normal genetic mechanisms, that we start to have problems. I hope that at least a little bit addresses your question. 

Another question? 
- My niece's father-in-law went to Thailand and was supposed to have stem cells, somehow, put into his heart because he had heart problems. My question is, is there real research being done on human beings or was this a scam? He did die. 
- Unfortunately, most of us will go on to not survive. That's a normal part of life. I'm sorry for the humor. If you go to the Web site that I gave you before about the clinical trials, those are international trials. As far as I know, no one is introducing cardiac stem cells into the heart in this country. In other countries, bone marrow stem cells are being injected into the heart. 

So it's possible that in Thailand there was a legitimate study, a clinical trial, that only could occur because it's not governed by US policies. We have very restrictive policies, which are probably best for us, though some people would argue that they're restrictive. I'm not going to state an opinion in that regard, because I'm not qualified to. But yes, there are studies, ongoing, in the world in different locations, where stem cells are being introduced into the heart. But you raise a valid point. There will always be someone to take advantage. So it behooves you before going to a place like that to get some information that legitimizes that particular circumstance. Because sometimes emotions run faster than logic.