106 Timothy Kamp

(applause) 

- Okay, thanks so much, Tom. And thanks everybody for coming out on a cool November evening. What I'd like to do today is just talk a little bit about stem cells and the heart and what that really has implications for in the way of regenerative medicine. So we'll start out a little bit by talking about the burden of heart disease. And from there, we'll go on to what are stem cells. From there, move on to how our understanding of cardiac biology has changed, how we can get stem cells to change in a dish to become heart cells. And then about cardiac regenerative medicine and the center here on campus. So we've got a bit to cover today, but I'd like it to be interactive. So if you have questions as we go, please feel free to ask them. I don't know. 

Tom, can you turn down the light in front so the screen is a little brighter, or is that impossible? Or could I do it, if I was smart enough? (inaudible) (laughter) While Tom is testing himself here, we'll go on. So I just want to start because I am a cardiologist here at UW. That's great. You don't have to turn it down too low. I am a cardiologist here on campus, and also have a basic research lab. The focus of my research is understanding heart disease and defining new treatments for it. And part of that is in education and making sure that everyone's aware of really how big a problem heart disease is. It remains the number one cause of death in the United States. 

About 71 million people in the U.S. live with one or more forms of cardiovascular disease. It causes about 37% of all deaths in this country. And it's estimated in direct costs for the U.S. economy to be about $403.1 billion. So this is a big public health problem. The majority of the problem here in this pie graph over here is related to coronary artery disease. That's the cholesterol building up in the coronary arteries. That can cause heart attacks, as well as a range of other cardiovascular diseases here. But tonight, I'll focus a little bit about coronary artery disease and what it means to have a heart attack and how cell therapy may help with that. On this slide, I just give you some images. These are MRI images that you may or may not be able to see here. 

I'll try and point out. Let's see, does that show up? That doesn't show up. So there's-- Well, you can't really see it. The walls are squeezing in in the heart in a normal heart, so I'll just have to describe it to you. All the walls come in in the pumping chamber of the heart the left ventricle, equally and symmetrically to eject the blood. And what happens with the heart attack is you lose part of that wall. Here's a heart that had a heart attack and there's a part of the wall not moving. Without a pointer, I can't really show you that. Maybe I'll use this. 

So this part of the heart, this black area is the wall. It's should be moving in, and it's not. And so part of the heart muscle stops working after a heart attack. And it becomes scar tissue. So the obvious question is, can we address that problem with new approaches to repair the injured heart muscle. So that's where the issue of stem cells come in. And first, I'll spend a little time describing what stem cells are and how we understand them. A simple definition of a stem cell indicates that it has the ability to divide or self-replicate for, really, indefinite periods of time, even throughout the lifetime of an organism. It can differentiate into various specialized cell types under the right conditions and it can potentially grow into very mature specialized cells, such as heart cells and nerve cells. There are a variety of different types of stem cells. 

Embryonic stem cells are one form. They're isolated from the pre-implantation blastocyst. That is after the sperm and egg unite to fertilize and form the zygote. That begins to divide. It forms the cystic structure called the blastocyst, which is sort of floating before it implants in the uterus. And if those inner cell mass cells are isolated from the blastocyst, they can then be cultured under very special conditions, originally on a feeder layer of cells that maintain them as undifferentiated non-specialized cells that are pluripotent, in that they can give rise to all the different cell types in the body. And they can grow indefinitely in culture. They can also be grown up as single cells into new cultures of cells. So that's what embryonic stem cells are. They were first isolated from the mouse. 

And mouse embryonic stem cells were described by Martin and Evans and Kaufman in 1981. And really set the groundwork for a revolution in biology with engineered mice models really helping us understand the function of individual genes by loss of function, knocking out a gene and seeing what happened to the mouse. Or gain a function experiments, adding a gene and seeing what happened to the mouse phenotype and the mouse cells. So this was a major advance for biology. And it's created a whole new tool for research. And even back in 1981, folks thought it would be potentially useful to have human embryonic stem cells. But as you can see, it wasn't easy to get the human embryonic stem cells. Jamie Thomson here was the first to make a step closer to that when he isolated embryonic stem cells from non-human primates from the rhesus and marmoset. And that was published in 1995 and 1996. But ultimately the interest really ramped up when Jamie successfully isolated embryonic stem cells from surplus In Vitro Fertilization embryos in 1998. 

That is embryos that were created for couples undergoing treatment for infertility, and in the process many embryos were created in a tissue culture dish. Some of which were implanted in the mother and went on to bear children. The rest were cryo-preserved indefinitely, potentially for future use. But with informed consent, Jamie was able to get these with the donor's consent, and isolate the embryonic stem cells. Here, a colony is shown growing on a fetal layer of mass embryonic fibroblasts, which has been a very powerful tool in this field. And the latest excitement, as Tom suggested is related to a new form of stem cell. This is something called induced pluripotent stem cells. And really, there have been several labs working hard on this. Perhaps one of the leading labs is from Japan. Yamanaka and colleagues started this work working in mouse and using mouse fibroblasts. 

Where they took fibroblasts, which is what we call a terminally differentiated cell. So we start out in development as a zygote, a very blank slate cell that then progressively specializes. And typically, our understanding of development is as this cell gets more and more specialized, it can't go backwards and get less specialized with normal development. And what this work showed is that if you expressed the appropriate combination of genes in very specialized mouse fibroblast cells, that you can reverse their clock and reprogram them so they become pluripotent. In other words, they can grow into all different cell types in the body, like embryonic stem cells. And the most recent proof that these really are pluripotent cells is that these induced pluripotent cells were injected into a mouse blastocyst in a study done by Yamanaka. And in that blastocyst, they incorporated and they became a chimeric mouse, shown schematically here after injection into the blastocyst. So you've got these cells growing into all the different cell types in the mouse. And then they can even get in the germ cells and they can do what's called going germline. So they can form mice from this single induced pluripotent stem cell. 

So they are truly pluripotent in the case of the mouse. And this was sort of a surprise. People didn't really think it would be that easy to turn the clock back on cells, to get them to behave like stem cells. And this is recent work, really starting to come out just last year. And the most recent paper was earlier this year. So the field is moving quickly because just this week we heard about human induced pluripotent cells that were isolated both by Jamie Thomson here and the Yamanaka group, where they expressed four genes using a particular way to transform the cells and get human cells into these induced pluripotent stem cells that may be important for regenerative medicine applications. The figure just shows that in Jamie Thomson's work, they were able to differentiate these cells into a variety of different lineages. When they formed teratoma tumor in mouse cells, they allowed the cells to spontaneously differentiate into neural-like structures, into cartilage-like structures, and into gut-like structures. Just a selection of different tissues that can form. Again, supporting the idea that these are truly pluripotent stem cells. 

And that's not to forget that there are also stem cells in adults, so-called adult stem cells, or progenitor cells, are a little more specialized. Probably the best description of adult stem cells comes from the blood system in the hematopoietic stem cell. It's present in our bone marrow, that then can specialize into what's called a progenitor that can give rise to a variety of different cell types. If it's a lymphoid progenitor, it can give rise to lymphocytes, specific blood cells. If it's a myeloid progenitor, it can give rise to other subsets of blood cells. And this is sort of the hierarchy of development of this blood system that's obviously quite important for us. And this has been, really, appreciated for a few decades, that the bone marrow contains stem cells that are able to self-renew. They undergo what's called asymmetrical division. So if this cell divides, one cell remains a stem cell, and the other starts to specialize a little bit. They are multipotent, but they're not pluripotent. 

That means they can differentiate into many cell types, but they can't differentiate into the full gamut of cell types present in the human. And the most rigorous test is you can take a single hematopoietic stem cell and reconstitute a blood system in an animal or a human. So really what the hematopoietic stem cells and more recent technology have given us are tools to find stem cells in other tissues. We've known about them in blood for a number of years. But only really in the last decade or so have we started to appreciate stem cells in other tissues. And that's because we've had tools that allow us to identify those cells. Otherwise, when we do the standard histology, looking at tissues, the stem cells don't really stand out as anything special. And it's only by using antibodies that recognize the various specialized proteins on those cells, as well as gene expression studies that we can tell that these are stem cells, as well as better techniques in microscopy to allow us to look at the tissues, and better ways to isolate the cells. And that's important because we'll go on and talk about how these tools have impacted cardiac biology. So now we'll start focusing a little bit on the heart and where I focus most of my effort and energy. 

And one of the dogmas of cardiac biology is understanding how the heart responds to injury. And a common injury is a heart attack or a myocardial infarction, as I tried to show you with the movie, which didn't work. But what happens with the myocardial infarction is a scar forms. And here is a section through a heart that's had a myocardial infarction. And the blue is the area of scar. All that blue tissue is dead scar tissue. They are fibroblasts, but there are no red muscle cells. So this part of the heart won't contract. So obviously, the larger that is, the bigger the problem is. It turns out this scar puts stress on the rest of the heart, causing it to sort of work overtime. 

And in the long haul, that causes the heart to do what we call remodel, get bigger, dilate, and go on to heart failure. So many forms of heart disease are associated with loss of functional contracting heart muscle cells. And so we typically thought that once you lose the cells, they're gone for good. And that was the dogma I grew up with in medical school and largely still the dogma being taught today. But when people have more recently been looking at things a little more closely, they found that things don't quite add up that way. For example, if you're very meticulous and count the number of heart cells in a rat heart, there are about 23 million myocytes. But if you also look at the rate that those myocytes are undergoing programmed cell death, which is called apoptosis, about 1.34 million cells die every month. So if you then take that into consideration and look at 20 months, you'd think there'd really be no heart left. But it turns out there are some fewer myocytes, but really not that many fewer. So how are we making up this difference? 

How are we making up for the cells lost via programmed cell death? Then others have looked for cell division in the heart. It's been very controversial, hard to find. But in diseased hearts, they can find some of those myocytes dividing. So maybe the heart isn't sort of fixed after you're born, and there is the ability to get new cells. Another clue along this line came with work earlier in 2000, where people did sort of interesting work in heart transplant patients. Something that modern medicine has created to help patients that really have no options with a failing heart. But it's also provided a research tool, and that is, if you transplant a female heart, females have two X chromosomes, into a male recipient, males have XY. And if you look at that female heart later on after it's been ingrafted, if the patient dies for some reason, and that's how they got these autopsy samples, you find out that in the female heart, it should be XX, there are Y chromosomes. Where did those Y chromosomes come from? 

Is there some population of cells getting into that heart that then are forming heart cells? So these have been sort of clues out there that maybe the heart is a little more complicated, and maybe it has some ability to repair itself. And at the same time, others have identified adult stem cells in a variety of tissues including skin, liver, fat, skeletal muscle, the GI tract, and so forth. So really, the breakthrough paper came pretty recently, 2003. We've been studying the heart for a long time. But just in 2003, really some definitive work was done that provided evidence that there are stem cells in heart tissue. So there is a population of adult stem cells, shown here as these small cells with a rim of green around them. I'm not sure if you can see that too well. But that green is identified with an antibody that recognizes a protein called c-kit, which is particularly present on stem cells. So these authors did a variety of other experiments to support the idea that these were stem cells in the heart. 

So this really is a fundamental change in our understanding of cardiac biology and gives us all sorts of new potential avenues to treat heart disease. So the good news is that there are cardiac stem or you could call them progenitor cells identified in the heart. They're self-renewing. In other words they can grow and they can grow from a single cell. They're clonogenic. And they're multipotent. That is, they can grow into heart muscle cells, but they can also grow into blood vessel cells. Having muscle without the blood vessels to supply it wouldn't be too useful. So these cells can do all that. So regenerate heart tissue, pretty encouraging. 

So the heart, like we also found out in the brain, has primarily terminally differentiated post-mitotic, so that means very specialized cells that can't divide anymore. But it's not simply only those cells. There's a hidden population of stem cells that have some ability to repair the heart. And potentially manipulating those endogenous stem cells holds promise for treatment for a variety of heart disease. And perhaps some of the most striking findings were produced by this work in zebrafish where Mark Keating and colleagues took a zebrafish and they snipped off the tip of the heart. They just cut it off and threw it out to see what would happen. And it turns out that a big scar formed there initially, a blood clot. But over time, that was replaced, and the heart totally regenerated in the zebrafish. So zebrafish can totally repair their heart after an insult like that. The bad news is we can't. 

So most of the insults that we are faced with in adult cardiology, stem cells that are endogenously present aren't able to take care of it, or we wouldn't be seeing it clinically. Why is that? It may be that adult stem cells become dysfunctional with aging. It could be in diseases like heart failure, the bone marrow-derived stem cells aren't working so well. And interestingly, many of the risk factors for atherosclerosis, in other words, a cholesterol build-up in the arteries are inversely correlated with the levels of circulating stem cells. In other words, if your cholesterol is high, and your bad cholesterol is particularly high, it knocks back the stem cells in the circulation. It's sort of a potentially a one-two punch. So it's nice to know we have stem cells in our hearts, but the way they are, they're really not taking care of many of the insults that we're faced with. So how can we get around that and treat that? And I think that's where the idea of harnessing the power of some of these stem cells and the tools of regenerative medicine will be helpful. 

Let me just talk about whether or not these stem cells can actually form heart muscle and some of the work we've done in our lab. And that work has looked at the differentiation or specialization of embryonic stem cells into cardio-myocytes, using a system where we take the undifferentiated cells and remove them from the specialized conditions that maintain them undifferentiated, form clumps of cells in suspension called embryoid bodies and then plate them down, and they spontaneously differentiate. We can then plate them on our little cover slips and look in there. And we can see contracting cells. I don't know if you can see that too well. But there are cells spontaneously contracting. They give us a not-too-subtle clue that heart cells have developed. They develop in sort of a random pattern. This embryoid body formation doesn't recapitulate formation of an embryo. Rather, it's a spontaneous process, and some of the embryoid bodies have these beating areas and some don't. 

So we've then gone on to study those using the tools-- (inaudible) I will do that. It's not important on this slide. (inaudible) I will do that on another slide. I can read it on my computer screen. (laughter) (inaudible) Tom would have to do that, because that's again, beyond me. But maybe he could. (inaudible) No, I'll fill in blanks where the red shows up. But on this slide, don't worry about it. So what this shows is a schematic diagram where we can take an electro physiologic apparatus, so an amplifier, and put an electrode, a micro-electrode, into the cells, and then measure their electrical activity. And why might we want to do that? 

One of the hallmarks of different types of heart cells is their electrical signature. Our heart is an electrical organ. The electrical wave is what triggers the contraction. So different parts of the heart have different electrical, what we call action potentials. So the pacemaker region of the heart, called the sinoatrial node, sort of has this slow upstroke action potential and this slow what we call depolarization before it. But that's quite different than action potentials in the atrium, the upper chambers of the heart, and the ventricles, the lower chambers of the heart, which have a much longer plateau phase. So the four chambers of our hearts have different types of action potentials. And that's sort of a nice signature, because it's made up of the opening, closing and activity of really, dozens, if not a hundred different electrical ion channels and transporters. It gives us sort of a fingerprint of the cell type. And so when we took our electrode and measured some of these action potentials, or electrical activity from the embryonic stem cells, we see that there are a variety of different electrical signals. 

There are nodal-like action potentials, suggesting that nodal-type cardio-myocytes can develop from the human embryonic stem cells. There are more atrial-like action potentials, more ventricular-like. So this is just a general proof of principle that the embryonic stem cells can form all different types of cardio-myocytes, which may be useful for different applications. If you've got a problem with a ventricle, you'd like to put ventricle cells in there. If you need a biopacemaker, you might want to think about more nodal-like cells, for example. Another reason that this might be important to have these stem cells beating in a dish is something to do with cardiac drug safety. Now I have to get you a little more into cardiology. This is a schematic of an electrocardiogram, and one QRS complex, which is essentially one heart beat. And it turns out that from the beginning of the ventricular activity to the end, which is called the QT interval, depends on how long the electrical action potential is in the ventricular muscle. So a longer QT means a longer action potential. 

And so we can measure an action potential in these heart cells. And we can see if a drug makes that action potential longer. So why am I telling you all this? It turns out that it's important, because there are certain potentially lethal heart rhythms that can be induced by certain drugs. Not as part of their planned effect, but as sort of a bad side effect. And this is an example of a patient actually admitted to UW Hospital, now more than a decade ago, who came in and was taking an antihistamine called Hismanal, and had fainted in church. And she got brought into the ICU, and they monitored her here with an electrocardiogram. Also put in an intra-arterial line so they could measure her blood pressure here. You can see the electrical activity of the heart here. And a long QT interval is here, which is a sign that the action potential is long. 

And what happens is a bad heart rhythm here that's called Torsades de Pointes. It goes very fast and you can see the blood pressure goes down to nothing. So if this wouldn't have spontaneously broken, this would have been a lethal event, if it didn't happen in the hospital. And that ultimately, as well as other studies, led to the withdrawal of this drug from the market. It's also led to the withdrawal of several other drugs, and is now screened for. With all new drugs coming through development, they're screened for this potential side effect. But there aren't great models to screen for that. So potentially using a cell like these embryonic stem cell-derived cardio-myocytes may provide a useful tool to help with drug safety, as well as other aspects of drug development. So now let's talk a little bit about cardiac regenerative medicine and where we are right now with that. So regenerative medicine, I think is an extremely exciting area. 

It's really largely new concepts. And it's rapidly growing. It explores, not only the use of stem cells and their derivatives to repair damaged tissues, but also looks at the ability to harness, for example, the body's own reparative properties using small molecules, for example, to turn on endogenous stem cells or to alter the ability of the body to repair itself. So it's a broad area that encompasses a lot of different approaches. But if we're going to think about cell therapy, we have to really take our time and think about the various steps to get from the research lab to patients. And the first step, really, is to get a well characterized cell population. And develop, if we're going to do tissue engineering, making pieces of tissue that will then implant into a patient. We have to really develop the appropriate materials to do that. And then test their properties In Vitro, that is in a cell culture dish. And then use them in animal models of disease, understanding how best to put them in the animal models to deliver them, understanding how to prevent the animals from rejecting them as foreign substances. 

Ultimately, we'd like to see in the animal models that they're effective and that they can ingraft, integrate into the host tissue. Improve the function of the host tissue and cause the disease to get better. But at the same time, equally and maybe more important, they have to be safe. We don't want to bring along infectious agents. We don't want to have any tumors formed. And we don't want any other potential problems, such as arrhythmias to come up. And then, ultimately these will get into human trials. So there's a bit of work to do before we get to patients with most of these cell-based applications. For cardiac cell repair, and many of the most prevalent forms of heart disease, are associated with a loss of function cardio-myocytes. So it is appealing to think about getting new cells in there. 

Whether it's coronary artery disease with a myocardial infarction, causing the loss of a big piece of the heart, heart failure with loss of functioning heart cells, certain arrhythmias are due do the loss of heart muscle. And maybe engineering new valves could take advantage of new cell therapies and tissue engineering. A variety of different cell sources have been considered for this. And many have already been tested, whether it's stem cells from the blood, so-called endothelial precursor cells, mesenchymal stem cells from skeletal muscle, from fat. And we've talked a little bit about embryonic stem cells, and also stem cells even from the heart itself. It's also important to think about your source of donor cells, whether they come from self, or whether they come from non-self sources. So we call that autologous if it's from self. So if you give some of your own bone marrow and they purify that to transplant into a heart. Or endothelial precursor cells are isolated from your blood, skeletal myoblasts from your muscle biopsy. Maybe what's on the forefront is the new induced pluripotent cells from a skin sample. 

Or cells isolated from your heart or cord blood cells. But maybe allogeneic cells will be a potentially important source. Right now, the embryonic stem cells represent an allogeneic source. Mesenchymal stem cells are another allogeneic source, or baby cord blood from other folks would be allogeneic or non-self source of cells. So again, probably hard to see here, and I'm sorry about that. This is a slide that really got us going down this road quickly. This is an experiment done, really, in early 2000 and 2001, by Peiro Anversa's lab and Don Orlic's lab, where they did sort of an interesting experiment. They took blood stem cells. So they isolated stem cells out of the bone marrow, isolated the stem cell population there. And it was from a mouse that expresses a protein called green fluorescent protein. 

So all those cells are GFP positive, which means they're green under the appropriate illumination. They transplanted them to a mouse that they tied off the coronary artery, so that mouse had a heart attack. Then they saw what those cells did. And here you barely can see, there are a bunch of green cells. But in the same range of tissue, there's also staining for a cardiac protein called myosin, suggesting those green cells, or those transplanted cells became heart tissue in the area of injury and are regrowing heart muscle. So that got everybody pretty excited to think that you could take bone marrow-derived stem cells and regenerate heart muscle. So that really got this field jump started. But it hasn't been without some controversy. A couple of other prominent labs have said that bone marrow stem cells do not regenerate infarcted myocardium. And this was, again, published in a fairly prominent journal, Nature, three years later. 

And so this has been an area of some controversy as to what the cells do. We, in our lab, used another cell population. And that is mouse embryonic stem cells, and transplanting them into a mouse heart model. And again, green cells have a reporter protein to tell us where the cells came from. And hard to see here, but this is an area of regenerated heart muscle. And the red is an antibody to what's called smooth muscle actin. So these are a couple of blood vessels you may be able to see. And this is regenerated blood vessel and heart tissue here. We think that transplanting embryonic stem cells can help the heart. And what we, and many other labs, have found is that transplanting these stem cells has a significant functional impact on the heart, in that if you look, these are what are called M-mode echocardiograms. 

What you see is after a heart attack, this is a particular measurement of how big the chamber is of the mouse heart. It's called the Left Ventricular End Diastolic dimension for the aficionados. It gets bigger after a heart attack. That is, the heart dilates after a heart attack, after myocardial infarction. And transplanting the stem cells blunt that, what we call remodeling of the heart. So the heart doesn't dilate as much after the heart attack. Likewise, the contraction of the heart, here shown as fractional shortening, gets much less, because you're losing functioning heart muscle. And if we transplanted the stem cells, that decrease in fractional shortening is blunted. So transplanting stem cells helps the heart, in that it blunts the remodeling and also improves the fractional shortening, or contractile properties of the heart. Two good things. 

How does it do it? Well, you know, originally we were thinking that it was all regenerational repair to help the tired heart. In other words, we formed new heart muscle. But over time, it's become clear that there are many, many potential mechanisms. And maybe these cells were only really secreting factors that helped the heart repair itself. Or maybe they're fusing with other cells in the heart to change their function. Maybe it's just stabilizing that scar so it doesn't keep stretching and putting the heart in mechanical disadvantage. Maybe it's turning on the cell cycle, in other words, getting those cells that we thought could never divide, to divide again. Changing the way that the connective tissue in the heart acts. Reducing that programmed cell death or drop out heart cells. 

Turning on endogenous stem cells. Forming new blood vessels. They're really many possibilities that are being investigated, that ultimately are helping the tired heart. It's really up in the air as to which are the most important mechanisms, and the most powerful. But I think this gives you an idea that there are many potential avenues that will develop over time to treat heart disease that are really fundamentally new insights. But it hasn't slowed us from getting into clinical trials. And these are, this is a list that I know no one will be able to read. And I just show it to you to show you how many trials there have been done since this discovery in 2001, up to 2006, these are the completed phase 1 and phase 2 trials looking at cell therapy in the heart. This is really unprecedented to sort of get some of these trials into patients, these cells into patients, at this quick tempo. And phase 1 and phase 2 trials are primarily designed to look at safety at end points, and less at efficacy. 

The phase 2 trials start to look at how beneficial this therapy is. So the conclusion from the initial trials was that as best we can see, putting primarily bone marrow cells into human hearts is safe. These are autologous cells, so they're taken from the patient, his or herself and put back into the patient, mostly after a myocardial infarction. But there's a variety here of different approaches here. It seems to be safe. Now the larger, the two larger trials that have been done to look and see if it's more than safe, if it actually helps. Here's a German trial called the Repair AMI Trial. They took patients after a myocardial infarction who had an impairment in the contraction of the heart. They transplanted these cells three to seven days after that infarction. And they looked at how well the contractile function of the heart improved after that. 

They saw, shown graphically here, that there was an improvement in the ejection fraction, or the contracting properties of the heart. And they also looked at clinical end points. In other words, how many patients died, had a recurrent myocardial infarction or needed to have a blood vessel opened again. That also was improved with the cell therapy. And now there's follow-up to this original study. It was published after one year of follow-up. At the meeting I was at earlier this month, now we're at two years follow-up. And these beneficial effects seem to persist. However, if you go a little further north to Norway, and you do apparently the same thing in a trial called the ASTAMI trial, where they randomized 100 patients after a myocardial infarction who had angioplasty, percutaneous intervention. Then they got these bone marrow cells on day six. 

They looked at left ventricular function by a wide variety of measurements. And the long story short is they didn't see any beneficial effect, either on ejection fraction or on clinical end points. So that has left us with some finger pointing going on, talking about whose cells are better than whose and how you isolate the cells. And so there is ongoing debate and a number of articles trying to understand how preparing the cells after you isolate them from the bone marrow may impact their biologic activity. I'm not going to go through all the details of some of these comparisons, but suffice it to say, there are some fundamental disagreements on the right way to prepare the cells, and whether that impacts-- Yes? 

- When you get a negative result, how do you know that the cells that you put into the heart are actually integrated in some way? 

- So the question is if you get a negative result, how do you even know you got the cells where you want them, and whether they're alive and functioning? So that's one of the fundamental challenges with human trials. It's hard to answer some of those very logical, basic biological questions. And I think, again, that's one of the reasons that animal studies are so important, really, to refine our understanding. 

The best we can tell is that when we put in these cells, we are targeting them pretty specifically, because in both of these trials, they put them in a catheter. And that catheter went into the coronary artery, where the infarction was. They blew up a balloon, so the cells couldn't go backwards out of the heart, but only could be shot down the coronary artery into the infarct bed. So they could only go into the tissue where there had previously been an infarction. So they should go that direction. The question is did they stay there? Do the stay alive? And we don't know from the human trials. There's no easy way to track that. In part, because one of the reasons this has been able to get to clinical trials so quickly is that if you take cells out of a patient, the patient's own cells, you just clean them up a little bit, and put them back in, there's less chance for things going wrong. 

If you take those cells out, mark them with a genetic approach so you can see where they are and get reporters, then you've monkeyed with them, you've got to do much more quality control. So for these first clinical trials, they're sort of the first gee-whiz approach. So your question is good, and we don't know. Yes? 

- Do you have baseline data on the changes in contractility without stem cells. 

- You know, I went over that. I glossed over that quickly. That's another great question. It turns out that even without stem cells, with optimum medical care, with medium sized heart attacks, ejection fraction tends to get better after the heart attack if you get on the right medicines and good medical care. What they're arguing is that there's an even greater improvement with the cell therapy. 

So they've compared it to placebo, which is squirting just the media the cells were growing in, to the cells. And both improved relative to right after the heart attack. But the improvement is even greater with the cells. Yes? 

- What was the change in ejection fraction? 

- It was not too large. You know, the absolute change was shown here about 2% to 2-1/2%. 

- What was the question? 

- Change in ejection fraction. 

- I'm sorry, so what is the change in ejection fraction? 

And the question I add to that is it clinically significant? So a normal ejection fraction in patients is generally greater than 50% or 60%. And so a 2-1/2% improvement on ejection fraction some people would argue is of borderline clinical significance. It's of statistical significance. In other words, it seems to be a clear difference. But will that impact clinical outcome? People have debated that. But in this trial, they did make some measurements of clinical end points, suggesting it could improve how patients did, at least in this trial, with their cells, in Germany. So there's a lot of question and debate as far as the right cell preparation and the right patient population, as well, that will maintain benefit. Some of the earlier trials studied patients that didn't have very big heart attacks. 

And some people have argued that if you're looking at patients with a small heart attack, they're prognosis, outlook, is very good. They're going to do well, regardless of cells or no cells. So it's sort of silly to study cells in those folks. So you really need patients that are in trouble, that have a significantly weakened heart, that are going to need some additional help. So only now are those patients being studied. Even though we're still trying to understand this biology, the trials continue. And we have been reluctant to get involved, initially. But now we couldn't hold back anymore. So we're participating in a trial here. This trial looks at patient population. 

It's a little different. These are folks that have intractable angina. So they have blockages in their coronary arteries that we can't fix with angioplasty, with the balloons, with stents, we can't treat with medicines any more. They have chronic pain, chronic symptoms with any activity. So they're really at the end of what we can do with medical therapy. Bypass surgery has usually been done, can't do it again. And so these are sometimes referred to as no-option patients. And so one of the hypotheses is if these cells can help grow new blood vessels, and get blood to the muscle that's not getting enough blood that you might improve the angina. You might improve the clinical status of these patients. So there's a trial that Amish Raval is the local principal investigator. 

It's a multi-centered trial. Douglas Losordo at Northwestern is the overall trial principal investigator. Where we isolate from blood CD34+ cells. This is a particular protein expressed on endothelial progenitor cells in the blood that we can then use magnetic beads, pull them out of the blood after they're mobilized using this apharesis technique. And then purify them. Then we take the patient to the catheterization lab and use a fancy technique of electroanatomical mapping called NOGA, where we can both look at the function of the heart, how it's squeezing, as well as the electrical activity, and identify regions of the heart that aren't squeezing so well but are still alive based on their electrical activity, which is a hallmark for that area of the heart not getting enough blood. That's shown here in this red area on this map. And the black dots are where we can inject cells. So you make multiple injections of cells in this area of heart muscle that's sort of alive, but barely alive, in the hopes of getting new blood vessels to form there and improving the patient's angina. That's an ongoing trial that's still enrolling patients. 

We obviously don't know the outcome of that. But I think it's important to give us a dose of reality as far as how long we can expect these developments to take, really, to get into mainstream medicine. And how much it's actually going to cost. I think it's useful to look at drug development as a comparison. So from getting an idea of a target for a drug until FDA approval of a drug, in the range of 10-15 years for most drugs. And in current dollars, costing in the range of $800 million per drug, on average. A pretty big investment in time and money. So thinking that we can get the complexity of cells into broad clinical applications over a remarkably short time period without much investment is probably not very realistic. Maybe cells are more like devices, such as the implantable cardiodefibrillator, ICD. That story, first described in 1969 in an animal experiment in Baltimore. 

It wasn't really put into a patient experimentally until 1980. And the first FDA approved application was in 1986. So there, 17 years, sort of from the proof of concept until patient application. What about stem cells themselves? Well the whole idea of stem cells isn't all that novel. Blood stem cells, or hematopoietic stem cells, were really first conceived of in the late 1940s and early '50s, largely based on our understanding of what happened after Hiroshima and Nagasaki. And the radiation sickness and loss various blood populations in those folks. And so the concept of a stem cell renewing blood in the bone marrow grew out of that. And the first successful bone marrow transplant was done in 1956, in identical twins, for leukemia. But it really wasn't successful in an unrelated donor until 1973. 

It's become more broadly available to patients, and there is now banking of bone marrow, and so forth, and it's a major clinical application. Again, that took from, really, the '50s until the '80s to get there. So I think there's a lot of promise with cellular therapies and regenerative medicine for the heart, but it's not going to happen tomorrow. Stem cells has also brought up questions of ethics and where the stem cells come from. This is largely focused on human embryonic stem cells since they're derived from the human blastocyst or embryo. And those embryos are destroyed. And so this raises questions that have challenged us over time. What is the definition of life? And other questions, such as cloning, also called somatic cell nuclear transfer. That's been suggested to be potentially a attractive way for what's called therapeutic cloning. 

Things like reproductive cloning are outlawed in most parts of the world. Also concerns about exploiting oocyte donors, or egg donors, to be able to generate these cell lines. And then questions such as chimeric animals. If you treat an animal brain with human brain cells, what have you really created there? If chimeric animals go germline and you have animal and human genes mixed in reproduction. A clear ethical concern. So that's led to a lot of discussion, thoughtful debate, and a lot of different opinions. Ultimately some guidelines have emerged from a variety of sources. Some of the best known are shown here. The National Academy of Sciences has produced guidelines for human embryonic stem cell research that you can look at. 

The International Society for Stem Cell Research has also provided guidelines. Both have suggested the formation of oversight committees, an ESCRO committee, which we have here at the University of Wisconsin, as well as other institutions have them. Standing for Embryonic Stem Cell Research Oversight Committee, to be sure that only ethically approved applications are underway at the university. And different countries have come up with different ways to provide oversight for this. And again, what happens in the United States, as far as our national policy. This is the Bush Administration policy, as per the Web sites. And it states that federal funds will be set aside to explore the promise and potential of stem cell research, with sort of the coda here that federal funds will not support the further destruction of embryos for ES cell research. So scientists must use existing lines, that were derived before August 9, 2001. So a compromise position by the Bush Administration. And the funding of more translational, that means getting from the basic research lab to the clinical arena is not well developed yet. 

And that's in contrast to drug development, right? The pharmaceutical industry has really developed this structure to get a compound that may be useful through clinical trials into patients. Many of the ideas come from basic researchers, but there's big cost, big funding that comes from the pharmaceutical industry. There is no such thing yet for cellular based therapy and regenerative based therapies. And so this is one of the growing pains that we have to work with right now with this novel approach. The federal policy has been a little complicated with the inability to use newly derived embryonic stem cells. So some of the states have seen fit to address this issue. And it's ranged from really trying to facilitate research using stem cells and embryonic stem cells. The biggest example is California, which passed Proposition 71, a couple years ago, that took $300 million a year of taxpayer money for ten years, or $3 billion, to support this research in the state of California. Obviously, they're pretty excited about it. 

And if you compare that to the amount of money the NIH spent in 2006 on stem cell research, $28 million, compared to the $300 million in California, you can see where the field is not quite level since this was spread throughout the country. Other states have provided initiatives to support stem cell research, while some states have passed legislation prohibiting it. So it's a varying playing field out there in the U.S. right now. To facilitate our work in stem cells and regenerative medicine here on campus, we formed a center recently. And our mission is, "To advance the science of stem cell biology and foster the clinical breakthroughs in regenerative medicine through faculty interactions, research support and education." And one of the things that we realize has been very lacking here on campus is a good way to communicate, both within campus and outside of campus. We really need to get better communication. That's important for you, too, because we have a Web site here that can provide some of the background, maybe I'll get it up here, maybe not. No, I've got to log in to the network. But we have a Web site that provides information for patients and also general information about stem cells and the researchers here on campus. 

We want to improve the basic research, work to translate this basic research to clinical treatments, understand the challenges in bioethics and also develop appropriate public policy that can really allow this new area of therapy to move forward. We want to get people working together across disciplines. Because as you can tell, this is getting complex. You know, we think of developing drugs as complex. That's a single molecule. If you think about the complexity of cells and really, the tens of thousands of different molecules working in sync there. It's not a simple process. So we want to get groups from chemistry, from engineering, the basic biologists, the clinicians, all working together. And also we're working hard to expand our educational activities here. So just a few things that hopefully you'll be able to take home. 

I have to say heart disease is still a big problem here. It's our number one killer. And we really are in desperate need of new therapies. Some of the new tools and approaches that have come through basic research have allowed us to understand that there actually are stem cells in the heart. And I think that's going to provide all sorts of new avenues for therapy. There are many types of stem cells that researchers have discovered and are working with: embryonic, different adult cells, stem cell sources, and now the newest kid on the block the induced pluripotent stem cells. These stem cells can self-renew or divide indefinitely in culture. They can form the highly-specialized cell types that are necessary to repair or regenerate a heart, or other organs, for that matter. And cardiac regenerative medicine is really a rapidly expanding field. We're developing new treatments. 

But it's a long road and it's going to be years before many of these treatments become mainstream. And I think this will revolutionize our treatment approaches for a broad variety of diseases over the coming decades. And all the work that I presented was the work of many folks working together. And I just show you some of their names. With that, I'll stop and try and answer any questions. (applause) Thank you. 

- You referred to embryonic. But there's also other options for stem cells... (inaudible) 

- The question is, I mentioned embryonic stem cells in the talk. And there are other options. 

So what makes embryonic stem cells attractive or not attractive. The embryonic stem cell, right now, is sort of the gold standard for a pluripotent stem cell. That is, it can form every cell type in the body. Most of the adult stem cells have a much more limited repertoire. So they can only form a certain collection of cells. Typically, if you take a stem cell from the heart or the brain, it's best at forming heart or brain. The other advantage of embryonic stem cells is that they're better able to grow in culture, to renew. So you can grow them potentially indefinitely in culture, which means you can grow up vast amounts of cells, which can grow into vast amounts of tissue. Whereas the adult stem cells undergo senescence. They stop dividing over time in culture. 

So you know, obviously, there's ranges for both of them. But the attraction with the embryonic stem cells has been that they're the most versatile cell, and if you grow up the most of them, and they do best in culture compared to the other cell sources. But you know, there are obviously limitations. You don't have unlimited numbers of embryonic stem cells. They're difficult to get because of the use of human embryos. So that takes careful consideration and the ethical concerns with that. So there are pluses and minuses. 

- How is it in... (inaudible) Response to these cells, which came from different individuals? 

- So that's one of the challenges if you're going to use these cell therapy. 

We talked a little bit about autologous, self-cells put back in and allogeneic. So the question was, does it matter, sorry, I'm repeating the question a little after the answer. But the question is how does this impact, if you use embryonic stem cells from one embryo that's not matched to the patient. And that is a concern. There is good reason to think that those cells would be rejected unless we get clever and find ways to allow those cells to be tolerized, so induce something that's called immune-tolerance. We know from work with solid organ transplantation, that is, kidney transplantation, heart transplantation, there's been ongoing research to understand how to induce that tolerance so you can take those cells. The easiest way is to give immunosuppressive drugs. And we know we've been able to do that with transplanted organs. That has some shortcomings, because the immunomodulating drugs can have some side effects that aren't too desirable. So I think it will take ways to modify the stem cells, perhaps to make them better able to be tolerated, as well as, for example, one strategy is well, if you're going to really do a lot of cell therapy and repair a heart in a patient, maybe you also need to give them a new bone marrow. 

So you can put in cells and reconstitute their immune system with bone marrow cells derived from that same embryonic stem cell line. There are many other sort of theoretical approaches. 

- Theoretically, your own cells would be preferential. 

- That's why there's a lot of excitement with these, you know, Jamie Thomson's recent discovery of these induced pluripotent cells. Then you don't have that immune rejection problem. And I think that is something that's really going to change the field. And has a lot of folks going that direction right now. The limitation is that those cells are genetically engineered with viruses that came in, to sort of get them back to stem cells. So the current generation, I don't think anybody would comfortable using those for therapies. Because where those viruses go in, they can alter genes and potentially lead to tumor or cancer formation. 

So this is a first step in the long-term, if we can find better ways to induce them. I think you're right, that would be very exciting. Yes? 

- You mentioned that the current cultured cells that you're creating can be used for drug reactions. Have any experiments been done where they've actually tried that out? 

- Well, I showed you one example there where the action potential got longer with the particular drug that's known to be an offender. So we have, in our lab, in the original publication, tested a few drugs and shown that in a small subset, it has some predictive value. Of course, the literature out there, there are thousands and tens of thousands of drugs, so how good this test is, we really need to go through a lot of drugs. And that hasn't been done yet to my knowledge. I think in the back. 

- With your last announcement, when I was listening on the national news and reading the national papers, it was always experts from Chicago or someplace else that were talking about what Jamie did. The University of Wisconsin was hardly mentioned. Didn't you guys feel slighted? 

- Well, you know, the question is... (laughter) If you didn't hear it, with the announcement of the induced pluripotent stem cells, the experts were not from Wisconsin, talking about it. And my own impression of that is, is that typically when discoveries are made, the media, especially big discoveries, like to get independent, outside experts that aren't tied in to the research. Of course if you talk to researchers at Wisconsin, they're going to tell you it's the greatest thing since sliced bread. So they want to find a somewhat independent, credible scientist somewhere else to discuss it. So I think that's driving a lot of those expert comments from around the world. And frankly, I'd rather be the institution developing it and having the discoveries made. 

Let everybody else talk about our work here. That's fine by me. Yes? I think up front. 

- Is anybody doing any work on stimulating the existing stem cells in the heart to do the process? 

- The question is, is anybody studying how to stimulate the endogenous stem cells in the heart to help repair the heart. People are looking at that, and are trying to add cytokines, or signaling molecules that are soluble, to turn on those. First we have to understand what turns on those cells, and we're still learning that. Then we have to be good enough to be able to see that they're turned on and what they do. So people are looking at that. 

And part of that is understanding what's called a niche. So where those cells live in the heart, and that specialized environment, and how to foster that. So people are looking at that. It's not close to clinic yet, though. Yes, another question in the front? 

- Yes, I thought I heard you say that NIH was spending $26 million on stem cell research. If one does the calculation, that's about what it costs for one hour in the war in Iraq. I wonder if you could comment on that. 

- So the question is the NIH is spending $28 million on stem cell research in the U.S. And that's roughly equivalent to the cost of one hour of funding for the Iraq War. And my comment on that is it wouldn't be my choice. 

But to comment more than that, I'm not in a position, unfortunately, to change it. I think over here, yes? 

- I believe you said earlier that stem cells are circulating. I was just curious, is there any natural event that takes place when a tissue is injured that generates a signal that says it's in need of stem cells? 

- That's a great question. The question is, stem cells are going around in the circulation. And is there a signal when there's damage to a particular tissue, come help me out. And I think there is. And those signals overlap what we've traditionally called signals for inflammation. So when you have a heart attack, your heart gets filled up with blood cells, white blood cells that in part chew up the damaged tissue. 

But it's probably also a signal to bring in some cells to try and help repair it. And it's the same with damage in other organs. There are signaling molecules that cause these cells to mobilize. We don't really understand them too well. And the real problem is that most of these cells are present in relatively low numbers. So you know, for example, a human heart has in the range of four billion muscle cells. So if you start doing the math, it's hard to get four billion stem cells into the heart. So you really are dealing with a very small number of cells with a massive task at hand. So there are ways to get those cells out, and those pathways are being dissected out. A question over here? 

- You talked about heart tissue. I wondered if you would just comment on clinical trials and successes or failures on regenerating tissues. 

- So the question is, I've had a cardiac-centric focus on my talk. And what's happening in other tissues. I think as many different tissues as there are, there are beginning explorations of clinical trials. There have been some trials in neuro diseases, primarily Parkinson's disease where fetal tissue is put in patients with Parkinson's disease. That was really done in the late '90s, and had equivocal results. But there are new trials with new sources of the dopaminergic neurons being considered. There's a trial here on campus using a human cell line that can form skin in burn patients. And in patients that have had bad burns that can't have grafts because they've been burned over 80% or 90% of their body, they've normally gotten cadaveric skin grafts. 

So Lynn Allen-Hoffman is leading a trial where she is taking a stem cell that she's growing in tissue culture that can form, they're called NIKS, Nearly Immortalized Kerotinocytes. And so they can form skin cells, and potentially be used for skin grafting. There are a variety of orthopedic applications where stem cells have been used, and tissue engineering for repairing bone. There's a variety of other things that are on the drawing board. One of the ones that I think is the most exciting is for diabetes. And diabetes, sort of the proof of principle experiments have been done, if we can get the right cells to do it for stem cell therapy. That is, as you may or may not know, juvenile onset diabetes is due to a loss of functional beta cells in the pancreas. So they are lost, so you can't secrete insulin. And so taking islets out of cadaveric donors, brain-dead patients, and isolating those beta cells and then injecting them into the liver, actually through the portal vein, in patients with diabetes, and immunosuppressing them, so they don't reject those cells, has successfully treated Type 1 diabetes and the patients have glucose control. You can take cells from an organ donor, isolate those cells, put them into a patient and potentially cure diabetes, with the caveat that you have to be on immunosuppressives, which is a problem. 

But to me, that tells me that if we continue to work with these stem cells, and can get stem cells that can form these beta cells, that's going to be a disease that may be amenable to cell based therapy in the not-too-distant future. So there's, you know, a vast array. I can't touch on them all, nor am I qualified to touch on them all. Yes? 

- What is the basic difference between stem cells and cancer cells? 

- That's a good question. The question is what's the difference between a stem cell and a cancer cell. I've told you stem cells are great. They keep growing forever in culture. What could be better? 

But I could've said, cancer cells, they're bad. They keep growing forever. And so they clearly have some overlap. Most of our stem cell populations are fairly quiescent in the body. That's one of the things that defines them. They don't really divide much until they're stimulated. They respond to the local environment in their ability to differentiate and form various specialized cell types. But cancer cells have sort of lost that check on their growth control. So they continue to divide. They don't turn off in response to the signals around them. 

There is an emerging concept of a cancer stem cell. So a cancer stem cell is that cell that's sort of slowly dividing that can give rise to the new cancer cells that rapidly grow and cause the problem. And some investigators believe that really, the failure of many cancers therapies is related to the inability to get rid of the cancer stem cell. It's sort of sitting there, hiding their in the background, and it's the fast dividing cancer cells we can get rid of. If you don't get rid of the cancer stem cell, the cancer comes back. So some of the biology clearly overlaps. Some of the gene regulation programs, some of the cell cycle programs, the ability of the cells to divide overlap. But their behavior is distinctly different. 

- Metastasized cancer cells sound like pluripotent stem cells in a way. 

- It does. 

So metastasizing cancer cells, are they like pluripotent stem cells going anywhere in the body? Probably not. These are cells that have some lineage commitment. In other words, if you've got lung cancer, these cells have some similarity to lung tissue, either an epithelial form or a small cell form, a different form. And when they metastasize, they still show that phenotype. So they don't go to the liver and become liver. If they were pluripotent cells and went to the liver and became more liver or repaired the heart, that'd be great. But they don't. They sort of are committed to that lineage. But they're abnormal in their growth control. 

They don't turn off when normal cells stop dividing. They do clearly overlap. Yes? 

- I know the brain is not your part of the body, but the brain controls most of us. (laughter) And how does that impact the rest of the stem cells that they're working on in the heart, whatever they're working on. 

- The question is how does the brain feedback on other stem cells in the body, and is there an overriding controller. I think there's no doubt that the signaling, that the nervous system impacts cardiac function in many ways. We know that from when you get excited, your heart rate goes up. And there are a variety of adrenaline like molecules that stimulate the heart that may impact stem cells. So there may be some relationship. 

I don't think in general, though, we think the brain has specific, strict oversight of all the stem cell populations in the body. At least our current understanding. But maybe it does. And as I always tell my neurologic colleagues, the brain doesn't matter if your heart stops. (laughter) I can't understand the brain, so I worked on the heart. Yes? 

- Do you think the field of epigenetics is going to have an impact on this work? 

- So the question is will the field of epigenetics have impact on this. And so obviously we have an educated audience here. But for the few that don't know the difference between genetics and epigenetics, standard genes, DNA, what we've sort of talk about all the time, we understand that our chromosomes in our cells have a DNA encoding specific genes that turn on and off, and create the proteins that are necessary for us to live. 

But epigenetics is sort of a different control mechanism. Sort of modifying the overall structure of those genes, so they can be turned on or off. And whether they're methylated, or other impacts on the gene, so they're available to turn on and off. And it's the epigenetics that really allows us all to commit to a particular lineage and not be able to go in other directions, to a large extent. So I think, especially for the induced pluripotent stem cells, epigenetics is a giant field which will be explored, and quite important. We really don't understand what's allowing certain parts of the genome to be active in certain cells. We're learning, and there's a lot of advances, but we're just starting to scratch the surface, so that will be important. In the back? (inaudible) So there was an investigator here on campus. Sorry, the question is what about stem cell research and multiple sclerosis. 

And Ian Duncan here on campus is in the Vet school, and actually studies the abilities of stem cells as a potential treatment for multiple sclerosis. Multiple sclerosis is an interesting disease in that it's what's called a demyelinating disease. So the axons, the particular parts of the nerves that send out distally, to go to the various parts of the body, are wrapped in a special sort of insulator called myelin. And with multiple sclerosis, they lose that myelin and become dysfunctional. So one idea is if you can get cells in there that would protect that myelin and re-myelinate them, you may be able to treat MS. That's ongoing work. I'm unaware of anything getting to clinical trials yet, but people are exploring in animal models the ability to treat MS. 

- Thank you very much. (applause)