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| Markus Grompe, MD |
Grompe is a professor of medical and molecular genetics at Oregon Health and Science University and also in the Department of Pediatrics. He studies metabolic liver diseases and the molecular genetics of Fanconi anemia. His research into hepatic gene therapy represents a potential cure for many hereditary and acquired liver diseases. Grompe is also working on methods to enrich for genetically transduced liver cells by in vivo selection and the use of liver stem cells.
Can you please define a stem cell?
A stem cell is a cell in the body that is responsible for renewing other tissues. It is not a differentiated functioning cell, but it is a cell that's sort of the reservoir for other cells that are needed in the body. Stem cells exist both before birth-prenatal stem cells-and they continue to exist in the adult organism.
An "embryonic" stem cell is actually a cell that doesn't naturally exist in humans or in animals. It's actually a kind of a laboratory stem cell that has been used extensively since the late 1980s for experimental biology. This very specific kind of cell [is] derived from early embryos and is used in the lab. Those are now available for human and mouse and a variety of other species. I tend to refer to the cells that naturally exist in the early developing organism as "prenatal" stem cells, because the term "embryonic" stem cell is already basically very narrowly defined as that particular kind of cell.
It all starts with the fertilized embryo when the sperm and the egg come together and that's the only cell really that one would describe as a totipotent stem cell, in the sense that that first one- or two- or four-celled embryo has the ability to give rise to the entire fetus as well as the placenta. So the difference between a totipotent and a multi- or pluripotent cell is that only the totipotent cells can give rise to both the placenta and the embryo both.
The embryonic stem cells that have been talked about so much are actually cells that can give rise to virtually all the tissues of the fetus and the adult organism, but they do not make placenta. So those embryonic stem cells that are talked about quite a bit in the newspapers and so forth, are not totipotent, they're not really little human beings. They have the capability to making all the tissues of the fetus, but not the placenta.
And the placenta is necessary for the fetus?
For complete development, yes. The very earliest cells are the ones with the most developmental potential. The embryonic stem cells that people are beginning to use for tissue repair studies and so forth are derived from the very early embryo at the so-called blastocyst stage, which is where the embryo has first developed a cavity within it and there's a group of cells in there called the inner cell mass. What people do for mouse and human and primate embryonic stem cells, is pick out those inner cell mass cells and then grow them in the laboratory. What's been learned from the mouse in particular is that you can actually culture these cells in a dish for many many generations and then inject them back into blastocysts and they have the capacity to develop back into a full adult mouse, which is the basis of a lot of the genetic manipulation of mice that we use in the laboratory.
I think that the distinction between totipotent and pluripotent is important in regards to the ethics of this discussion, because some people are under the impression that embryonic stem cells are little people being grown in the tissue culture dish in the lab-these are not embryos. They're embryo-derived. So in the process of generating embryonic stem cells an embryo is destroyed, but it's not the same thing as cloning or actually having the equivalent to a human conceptus in the lab.
Because there has been controversy about this and there are ethical questions, are there some labs that have been using the totipotent cells? Is there advantage to doing research with that and has it happened?
Well, there are labs that work with actual human embryos. Those are particularly-in terms of the research applications-the people interested in cloning. Those cells actually grow very very poorly and so you really don't have the ability to propagate them extensively and use them for tissue repair studies. What people have been using them for is to basically try to put into these embryos the nuclei from adult cells and that's called cloning.
That's what we've been hearing about here the last few weeks or months, is basically killing a human embryo and transferring the nucleus of an adult cell into it. I think the ethics with embryonic stem cells come from the fact that to get them you have to destroy an embryo. But once they're there and exist and can be grown, there are no further ethical problems at that point.
I think this is where the policy of the Bush government has come from, is the fact that [it is OK to use] the already existing lines.
So the ethical question for using the pluripotent cells arises because the embryo at that early early stage is essentially destroyed?
Yes. So, basically, to get embryonic stem cells, the embryo is allowed to progress to the blastocyst stage, which is 32 or 64 cells, and is harvested essentially at that point, or a portion of the embryo is cut out. Basically that's the end of that embryo except for the cells in the inner cell mass that can then essentially grow indefinitely, they're in a sense immortal.
Now can you talk about adult stem cells?
Adult stem cells are, for the most part, tissue-specific adult stem cells; meaning that for a certain tissue, you need in some instances a stem cell that continuously divides and spawns new cells to stay alive. There are tissues that turn over all the time and you basically need stem cells, so that would be blood, skin, intestine, or sperm production. Those are all examples of tissues where there's continuous cell division going on and all of those tissues have stem cells that are responsible for tissue renewal.
People have known and written about [these] for a considerable length of time. It's now become apparent that most, if not all, tissues actually have stem cells. Although most tissues are not continually dividing all the time, there are cells that are responsible for tissue repair in the case of injury. So for example, let's say your liver gets hurt by a virus or something like that. Even though liver cells on average only divide once a year in a normal person, you can wipe out your liver with a virus or an injury quite drastically and then you might need stem cells. These are adult stem cells that we call facultative, meaning they are only activated when they're needed as opposed to those in the tissues like blood that are always activated and continuously dividing.
Finally there's a new concept in terms of adult stem cells, which is the idea that adult organisms, including people, continue to harbor some of these very early stem cells like the embryonic stem cells, basically pluripotent stem cells. That's really a concept that's only emerged in the last five, six years and has generated a lot of excitement in terms of the therapeutic potential of those cells.
Can you describe the process that takes place that turns a totipotent cell into a pluripotent cell?
The human zygote is a totipotent cell. It basically can give rise to all the adult tissues, all the fetal tissues, and so forth. It seems that as the number of cells increases, the developmental potential becomes further and further restricted.
The way a totipotent cell, which is the very first cell at conception, turns into pluripotent and multipotent cells and so forth down the line, has to do with the phenomenon of developmental restriction. Obviously, the adult human body consists of many many different tissues. Actually people have estimated that there are more than 200 different cell types that make up the adult organism. In adult organisms, it's kind of important that one tissue type doesn't turn into another. For example, you don't want your liver cells turning into neurons, just without any reason to do so.
So basically development is typically a hierarchy starting from an undifferentiated primitive cell that becomes more and more patterned or what we call differentiated as it develops. In the extreme there are cells that we call the "terminally differentiated" cells, like the epidermis-those cells are terminally differentiated, they will never turn into anything else. But they are derived from a stem cell that lives in the epidermis down at the bottom. That stem cell gives rise as far as we know really only to skin. But in development it came from another stem cell that could give rise to all sorts of ectoderm, including the kind of cells that give rise to teeth and hair and so forth. So basically you start with a totipotent cell and as the number of cells increases, their developmental potential becomes increasingly restricted. In the adult organism there are lots and lots of cells that can be only one thing and very few cells that have multiple potential.
What are the advantages of using embryonic stem cells or prenatal stem cells for research?
The reasons embryonic stem cells are advantageous for research are many-fold. The main advantage that I see in terms of practical use is the fact that these cells can grow virtually indefinitely in the tissue culture dish. It's possible to share cells between laboratories, it's possible to have comparison of results between different laboratories. But just the sheer fact that they are what we would call immortal allows very very easy experimental manipulation of the cells.
Embryonic stem cells have the other advantage that they are multipotent and can turn into many different types of tissue in culture and therefore it's possible to learn from studying embryonic stem cells how differentiation occurs. Basically, how you go from multipotent to developmentally restricted and finally differentiated. People are using them for that purpose.
What's the process to obtaining embryonic stem cells?
The procedure by which embryonic stem cells are made differs a little bit from species to species, but basically fertilized embryos are taken in the tissue culture dish and they are allowed to develop to the blastocyst stage, which consists of 32 to 64 cells. You can see that under the microscope, it takes several days for that to happen. At the blastocyst stage there will be a group of cells on the inside of the embryo called the inner cell mass. They are dissected out under the microscope and then they are dispersed and grown on a group of cells that we call feeder cells. They essentially then start to grow like bacteria, doubling and doubling and doubling again with virtually unlimited capacity for that process.
The nice thing about embryonic stem cells is, though, they haven't forgotten how to go back and be differentiated and stop growing. So that if they are put back into an environment such as a developing embryo where they get the right signals, they stop growing uncontrolled and they start behaving like a proper inner cell mass again.
Are there advantages of using adult stem cells for the same type of research?
The adult stem cells have the advantage of the fact that you can have many more cell donors, it's easy to get the cells. For example, it should at least in theory be possible to get adult stem cells from any person who wishes to donate cells. In terms of research, they have really not many advantages over embryonic stem cells, with the possible exception that what we learn about differentiation from embryonic stem cells could potentially only pertain to embryonic differentiation.
There's a paradigm in developmental biology that says that the way a liver stem cell becomes a hepatocyte, for example, in the embryo, is the same process in principle that happens later in life when a liver stem cell becomes a hepatocyte. But that is only a hypothesis at this point. We really don't know in most cases whether adult stem cell differentiation mimics embryonic stem cell differentiation. It's possible that what you would learn from embryonic stem cell differentiation would not apply to adult stem cells.
And therefore it does make sense to be doing some of the same research with both kinds?
It makes sense to compare embryonic stem cells with adult stem cells because the answer may not be the same in both cases.
Where and how do you obtain adult stem cells?
Adult stem cells that are multipotent have really only been described from one source so far, and that is from the adult bone marrow. A group at the University of Minnesota has described a group of cells called multipotent adult progenitor cells, and they in many ways behave like embryonic stem cells. But that's to our knowledge, the only source of multipotent stem cells in the adult so far. But there are also of course many tissue-restricted adult stem cells, such as the blood stem cell or the intestinal stem cell, and people are studying those individually as well.
And those cells would be considered multi-potent because they really only can create what they're taken from?
Yes. Those cells would be considered multipotent.
Can you summarize in a "compare and contrast" fashion the use of embryonic versus adult stem cells?
Embryonic stem cells and adult stem cells can be both used for research. There are several advantages and disadvantages to both. Again, the most important advantage of embryonic stem cells is that they can be easily grown to large numbers, it takes a very short period of time to grow a lot of them, which makes it easy to study. Also the fact that they can develop into multiple tissues makes it possible to study those processes.
The disadvantage of adult stem cells in comparison is that they grow much more slowly and at lower density, so it takes a long time to make lots of adult stem cells. However, the advantage of using the multipotent adult progenitor cells is that they would potentially teach you about developmental processes in the adult, which could be very different from embryonic stem cells. So, in that sense, even though they grow in a more difficult system, they have the advantage of probably reflecting the status of adult stem cells more readily.
In terms of clinical application, there are also quite important differences between embryonic stem cells and adult progenitor cells. The most important one is that embryonic stem cells are derived from donor fetuses that are immunologically mismatched to the person you'd want to be treating. You would have the same issues as you currently have with organ transplantation or with blood transfusions, where you might have to actually use immunosuppression to be able to use those cells. With the adult stem cells it should be possible to derive those cells from the patient herself, so you at least in theory may be able to get away completely from using immune suppression. So the immunological mismatch is a problem.
The other issue with embryonic stem cells is that when they're injected or when they're transplanted without additional modification, in animals at least they form tumors very easily. There is a significant safety issue with embryonic stem cells. Whereas the adult stem cells as they've been studied to date, do not form tumors in any kind of setting. So there are differences for clinical use in that regard.
Would another disadvantages of using adult stem cells for clinical use be that the genetic makeup of that cell is going to be the same as the person you're putting it back into and the same mutations may be there?
Adult stem cells are thought by some to have the disadvantage of being a problem with genetic disease. For example, if you derive adult stem cells from a person with a lung genetic disease, the cells that you derive from that person are also of course going to have that same genetic disorder. Even though they're immunologically matched, you couldn't do a cure with them because they have the same genetic defect. However, that problem is easily surmounted, because like embryonic stem cells, adult stem cells grow extensively in tissue culture. It is possible to correct the genetic defect first in vitro and then use those genetically modified cells. The only difference between the stem cells and the patient are that the stem cells have been cured of the genetic defect.
You have referred to the bone marrow cell as being pluripotent as opposed to multipotent. Are there specific examples in which someone has succeeded in culturing a different kind of tissue, like a liver tissue, from the bone marrow tissue?
Yes. The bone marrow is a rich source of a variety of stem cells. First we have the original hematopoietic stem cell, the precursor to all blood lineages. It's been known about for a long time. More recently there is another kind of stem cell that's been discovered in the bone marrow called the mesenchymal stem cell. It can be used to make mesenchymal tissues, which are muscle cartilage, bone tendons, and fat. Finally, there are now the multipotent bone marrow cells, which are different from the two previous ones, which have been shown at least in tissue culture to differentiate into epithelial cells, including liver cells, neurons, muscle cells.
Virtually every cell type that has been seen in embryonic stem cell differentiation has also been seen with these multipotent adult progenitor cells in tissue culture. In 2002, they also showed that these cells injected in vivo in animals or early embryos behave very similar to embryonic stem cells. So, the multipotent adult progenitor cells have virtually the same developmental potential in tissue culture, but also when injected into blastocyst as the classic embryonic stem cell.
The only example of a totipotent stem cell is the fertilized embryo at the early stages, basically up to the eight or so cell stage where you could split up the embryo and get eight identical twins. That would be totipotent. There's really not a clear distinction between pluripotent or multipotent. Basically what that means is that that particular stem cell can give rise to multiple different cell types, but it's not defined further than that. An example of a multipotent stem cell would be the hematopoietic stem cell, which can give rise to all the blood lineages, including white blood cells, leukocytes, lymphocytes, the platelets, the red blood cells, and macrophages. They all come from one single stem cell that would be called multipotent.
Can you talk about the stem cell research that has happened in regards to diabetes?
Diabetes Type I-juvenile or childhood diabetes-is characterized by a loss of the insulin-producing beta cells in the pancreas. Basically a person with Type I diabetes has no beta cells left. And unfortunately, the adult pancreas, or the post-natal pancreas, does not make new beta cells if the old beta cells have been destroyed. For that reason, people have been looking for a way to make insulin-producing beta cells from other cell types.
For many years people have been interested in whether the adult pancreas itself has some cells that can become insulin-producing cells and can be coached down that direction. Even though the body doesn't do it itself, you could potentially make it happen in the lab, and those are the so-called pancreatic stem cells that many laboratories are still interested in. That work, however, has not gone very well and we still don't have cells like that.
However, since about two years or so it's become apparent that embryonic stem cells can be turned into insulin-producing cells in tissue culture. So a lot of hope now rides on the concept that you could take these embryonic stem cells we've been talking about and educate them in a step-wise fashion to become insulin-producing beta cells. The proof that this can be done in principle has already been achieved in the mouse, where it's been possible.
Can you talk about the pros and cons of a patient receiving his or her own cells?
There are disadvantages and advantages to using the patient's own cells for the treatment of Type I diabetes in terms of stem cell therapy. It's important to note that Type I diabetes comes about by immunological rejection of the patient's own beta cells. So, the thinking is that if you were to generate beta cells from that patient's own tissue, you would basically restart the entire process. In other words, the immune system would come after those beta cells again unless you use immune suppression. If you use embryonic stem cells from a donor that's not tissue matched to the person with diabetes, it could have both a positive or a negative effect. The negative effect would be that the immune system might also attack those cells because they are foreign. The positive effect might be that that immunological rejection may be more easily managed than the original immune rejection of the beta cells.
Until those kind of experiments or studies are done, it's really impossible to know whether that would be an advantage or disadvantage to use tissue matched or non-matched cells. But the immune rejection is going to be an issue with Type I diabetes whether you use the patient's own cells or not.
Can you talk more about the ethical issues in this area of research?
The ethical concern about human embryonic stem cells comes from the fact that to generate embryonic stem cell lines means that you have to take a human embryo and harvest it, in other words, kill it. The cells of the embryo are dissociated at the blastocyst stage and then grown as a cell line from there on. But if you were to take that same embryo and implant it into the placenta, it would develop into a baby. So, basically, to get an embryonic stem cell line means the death of an embryo. And the ethical debate boils down to when does human life start. Is it at conception with the potential to develop into a full human being or at some later stage? Obviously people do not agree on that particular point.
What are your personal big unanswered questions in regards to your research?
My primary interest in stem cell research lies in the therapeutic use of these cells. What I see as the current main interest of the field is how to get multi-potent embryonic stem cells to become differentiated cells. That's certainly very important, but it only gets us half way to therapy. The second really really important component to using cells for therapy is to actually get them into the patient. And that is currently actually maybe a more difficult step than the actual isolation and procurement of the cells.
So, to give you an example, in order for a bone marrow transplantation to work, bone marrow transplantation is a form of stem cell therapy. f you just had the hematopoietic stem cells and transplanted them into a person with a blood disease, nothing would happen. In order for the stem cell to become clinically relevant, you have to find a way to give the transplanted cells a growth advantage so that they take over. In the case of bone marrow transplantation, we've learned to do that. Basically what one needs to do is to give the patient lethal irradiation and chemotherapy to make space for the therapeutic cells. Now, this is also going to be the case for all other cell types that are going to be used therapeutically. Many people are under the impression that just transplanting stem cells is somehow miraculously going to result in a cure.
What I'm convinced of is that we need to learn how to get these stem cells to grow better than the endogenous cells upon transplantation. Our own particular work is focused on the liver, where we have identified the way in animal models how this can be achieved. So, my lab's interest is primarily in how to get repopulation to work as opposed to how we get the cells. In many cases we already have the cells, but using them therapeutically requires that we are able to isolate them and get them to repopulate an organ. Once we figure out how to do the equivalent of lethal irradiation and chemotherapy, for example for the liver or the kidney, then I think many many different cell types will work, including embryonic stem cell-derived cells, adult progenitor cells.
It's not the cell-I like to say in my lab-it's how you get it to grow. So that's the challenge I'm interested in.