This used to be kind of the norm in the 70s and 80s, but I would say now he is one of the very few remaining that still participate in basic research and active clinically and it is a pleasure to hear you speak. Thank you.
Speaker 1: Thank you for that kind introduction and I would like to thank the organizers for inviting me here today and allowing me to talk about the subject. I am going to talk to you today about stem cells and endometriosis and more generally stem cells and the uterus. We have learned a lot in the last few years about the role of stem cells in the reproductive tract and as you will see, there are some good things that come out of having stem cells in the reproductive tract but also some negatives and endometriosis being the primary negative. So, first what are stem cells? I think most of you probably have some familiarity with the concept that stem cells are these undifferentiated cells that are capable of not only self-renewal but giving rise to other more differentiated cell types. You know, most normal cells when they divide, give rise to two identical cells. They just replicate themselves. Stem cell would divide asymmetrically. So, it preserves the stem cell and gives rise to another cell that can go on to differentiate often into multiple different cell types. So, these cells can be obtained from the embryo, embryonic stem cells, and we know the early embryo gives rise to a whole human being. So, those stem cells are thought to be todipotent. They can give rise to every organ in the human being and they can also be found in adults. We now know that there are stem cells in many, if not, all organs that can be used to generate and repopulate those organs in the setting of injury or damage. These adult stem cells again remain relatively undifferentiated and can give rise to several of the cell types in a particular organ. So, adult stem cells probably arose very early in development. They travel to a specialized _____ capability and then they give rise to the tissue in the surrounding organ and again help it respond to damage and repair an injury or normally replace that tissue. When there are local stem cells like this, we often call them progenerative stem cells. They give rise to the, again, surrounding tissue, but there are also stem cells that are found in particular areas that can travel to organs in other parts of the body and regenerate these organs, repair these organs; bone marrow is one particularly rich source of stem cells.
Well, the endometrium, it should be no surprise to any of us has progenerative stem cells that we know we shed in each menstrual cycle, the endometrium every month. You have to regenerate that endometrium. It is one of the tissues with the highest turnover. So, it is no surprise that there are stem cells in the uterus that give rise to a new endometrium in each monthly menstrual cycle. I showed here some work from Caroline Gargett’s Group in Australia that identified some of these progenerative stem cells often in the basalis layer or lower layer of the endometrium that is not shed with menses and that layer we have known for many years gives rise to a whole new endometrium. So, it is not surprising that there are stem cells there that can give rise to the different cell types we find in the endometrium, both glandular and stromal cells, and it is thought that endometriosis that occurs through retrograde menstruation is really shedding of these progenerative stem cells.
It is these cells that have this tremendous capability to divide and regenerate the endometrium that when shed are most likely to give rise to endometriosis if they travel in a retrograde fashion. So, it is probably stem cells involved in the routine endometriosis that we most commonly see from retrograde menstruation. Some of the work we have done over the last few years have shown that these stem cells can come from areas outside of the uterus including the bone marrow. Now, the bone marrow has been shown to have these mesenchymal stem cells that have tremendous potential to differentiate into multiple different organ systems including some of the things shown here; muscle, fat, liver. We reasoned that if bone marrow stem cells can give rise to all of these tissues that do not have the turnover that endometrium does, of course they would be likely to give rise to endometrium as well, a tissue that has to be regenerated and replaced in every menstrual cycle. So, we ask the very simple question, can bone marrow stem cells differentiate into endometrium and we did a very simple initial experiment. We just looked at four women who had had bone marrow transplants and we looked at women who had had a single antigen mismatch. So, these were not a perfect match and each of these women had an HLA mismatch that we could use to identify the origin of the cell that it come from the bone marrow transplant or from that women’s own endogenous cells. We looked at reproductive age women and we looked at women who had had chemotherapy and total body radiation and then bone marrow transplant and here I am going to show you, I would not go into all the details but this is looking at the mismatch HLA antigen in the endometrium of somebody who would have had a bone marrow transplant and sure enough, these little bands here correspond to the mismatch HLA antigen that we started to see in the endometrium of women who had had bone marrow transplant. Some of the cells in their uterus came from bone marrow, not from their own endogenous endometrial cells. Well, that should not be totally surprising because after a bone marrow transplant, all of your white blood cells are of bone marrow origin and we know that white blood cells migrate into the endometrium. So, we started to look specifically at what these cells were and here we stain the brown staining as looking at the mismatch HLA antigen. So, here is an endometrial biopsy from somebody who had had a bone marrow transplant and the brown cells that you can see here are cells that have the HLA antigen of the bone marrow donor, not that women’s own HLA antigen. These are cells that arose from stem cells and they do not look like white blood cells that we expect to migrate in and out of the uterus. These clearly look like endometrial epithelial cells, the blue or the not staining for the mismatch HLA antigen, so those are the endogenous endometrial cells that the women had prior to bone marrow transplant.
We looked at the stroma. Here, we did some serial staining just to be sure that these were not white blood cells. The blue do not stand for either the mismatch HLA antigen or the marker of white blood cells we used here called CD45. So, these are her own endogenous endometrial stromal cells. These cells that are staining in red here are staining for a white blood cell marker. So, there are white cells coming from bone marrow as we would expect that migrate in and out of the uterus during the menstrual cycle, but these cells that the arrows point to are not white blood cells, but they do stand for that mismatch HLA antigen. So, clearly showing that these are from bone marrow origin, but these are endometrial stromal cells. They are not migratory white blood cells.
And here just some data that showed that these cells that have a mismatch HLA antigen also express several of the markers of mature endometrium. They not only look like endometrial cells, but they are expressing the genes and proteins that are necessary to make them function as normal endometrial cells. We also developed a mouse model to see if we could recapitulate this. In our initial studies, we looked at only women who had had bone marrow transplants from women. So, men may not have stem cells that could generate a uterus or endometrium. Well, in reality here, we are going to show you some data where we used male mice for the bone marrow transplant. Even men have the potential to generate endometrial cells in their bone marrow. So, it must be some very undifferentiated cells in the males that can even give rise to endometrial cells. First thing we did again after the bone marrow transplant, we looked at some Y chromosome markers, that is, just PCR and we can see that this is transplanted from male into female, we can get the expression of the SRY genes something on the Y chromosome that female should not express. When we transplant female to female, we do not get that and here is a male who has a control. Then, we looked at with Y chromosome FISH fluorescent in situ hybridization for the Y chromosome in the uterus of these mice. Here is a male as a control. Lots of these little red dots are signaling the Y chromosome. Here is a mouse that we did a female to female bone marrow transplant, we do not see any of those red dots. Here is the mouse that had a male to female bone marrow transplant. This is the lumen of the uterus. So, this would be the endometrial epithelium right here, these two rows of cells and there is a red dot there that is the Y chromosome. So, there is a male cell that came from the bone marrow that is incorporated into this endometrial epithelium and here is a stromal cell showing the same thing.
We also looked at some other markers again just to make sure we really were confident of our findings. We looked here again. It is a male control. Here we looked at the marker of white blood cells, CD45, and that green is showing you that that is a white blood cell and it has a little red dot in the center, so it is from the bone marrow as you would expect after bone marrow transplant, all white cells should be from bone marrow donor. But, here is again the lumen of the uterus in this mouse. Here are the epithelial cells here, here is a cell that again has a Y chromosome, so it is a cell from the bone marrow. It does not have that green color, the CD45. It is not a white blood cell and it expresses the yellow marker here of cytokeratin, which is a marker of epithelial cells. So, it shows that this really is an endometrial epithelial cell that differentiated from bone marrow-derived stem cell but the question that brings us all together here today is can stem cells contribute to disease, in this case endometriosis, and we again went through our animal model to ask could stem cells contribute to that by feeding the endometriosis? I think, you know, we know there are lots of theories for endometriosis that retrograde menstruation is the most common one or Sampson’s theory. There are embryonic rest, there is metaplasia, there is clearly some immune dysfunction in some women with endometriosis, there is a genetic predisposition in some women for development of endometriosis, but I think stem cell origin of endometriosis now has to be added to this list as a new theory for the etiology and I will show you some of our data in a moment and in Sampson’s theory, probably I am not discounting Sampson’s theory. Clearly, retrograde menstruation does account for some forms of endometriosis and there is good evidence to suspect that that is true that we usually see endometriosis in the dependent portions of the pelvis where we would expect gravity to carry these cells. We often see retrograde menstruation, and essentially all women have at least some retrograde menstruation; if we block the outflow tract, if we block the cervix, so that menstruation flows backwards, we almost inevitably develop endometriosis.
So, clearly retrograde menstruation can cause endometriosis but Sampson’s theory does not account for endometriosis that is outside of the peritoneal cavity or those rare reports of endometriosis in men, where does that comes from?, clearly not from the uterus. So, could stem cells be contributing? So, we looked at our animal model to ask if stem cells could contribute to endometriosis and here what we did is we created endometriosis by taking endometrial cells from the uterus of a certain genetic strain of mice that have a particular color marker we could look at, lacZ transgenic mice, and from normal wild-type mice and we sutured the wild-type endometrium into the peritoneal cavity of these lacZ transgenic mice and we removed their uterus. So, there was no way that any cells turning up in this endometriosis could have come from the uterus whether it is retrograde menstruation, whether it is lymphatic or hematogenous spread, and these cells had to have come from a stem cell source. Here just to set this up for you, here is what a lacZ transgenic mice would look like when we stain for an antibody for this gene, it turns it brown, wild-type controls do not turn brown. Here is where we sutured this endometrium into the peritoneal cavity, this is the wild-type, it is not brown, you can see the line of demarcation where the cells below this line are brown in the lacZ transgenic mouse, and here is what we found several weeks later that there were cells in that endometriosis that were derived from the host animal, remember that host animal did not have a uterus. This could not be cells coming from endometrium. These are cells that are coming from stem cells from another source whether it would be bone marrow or perhaps even other potential sources of endometrial-derived stem cells. Just to make sure it was not an artifact of the antibody we were using, we also looked at beta-glucosidase activity, which is something that would also light up those cells that came from the stem cells and here we can see both glands and stroma that arise from the stem cells.
So, I think this is a novel origin of endometriosis and really probably a novel mechanism of disease in general, ectopic differentiation of stem cells, cells forming new differentiated cell types in the wrong place, may be also contributing to other disease, but clearly contributes to endometriosis in this case, probably more than just a novel theory of endometriosis, a novel origin of disease. An endometriosis probably is not just one disease. As I mentioned, I do not want to discount retrograde menstruation. I think it probably plays an important role in most endometriosis that we see, but there probably are forms of endometriosis that do not arise from simple retrograde menstruation. Metaplasia may be playing a bigger role in endometriomas. Stem cells probably are the origin of endometriosis outside of the peritoneal cavity in the lung and the brain and other areas that we occasionally find endometriosis and in addition, we believe that they still continue to feed and propagate the lesions in the peritoneal cavity as they clearly did in our mouse model. In the couple of minutes that I have left, I am showing you the sort of the bad side of stem cells in the uterus. If I can take a minute to just talk to you about potentially the good side of having another source of stem cells, maybe stem cells in the uterus might be a readily accessible source that we can exploit for other purposes in regenerative medicine to repair other organ systems. Just to tell you very quickly about some work we have done in stem cells trying to generate different cell types from endometrial stem cells, so we could get an endometrial biopsy from a woman, use various techniques that we can use to coax these cells to differentiate down different pathways, and see if we can come up with some new cell types that might be beneficial so that stem cells here will be used for more than just generation of endometriosis, but potentially every woman could have an easily obtainable source of her own immunologically-matched stem cells that we can easily regenerate in each menstrual cycle. Here, we used a media and a technique that would allow these cells to differentiate into chondrocytes of the cells that make up cartilage and this is our control and these are the cells that we treated with certain media. I do not have time to go through all the steps and its thought, but this is a chondrocyte. We can generate cartilage. Here, we could generate neurons from endometrial stem cells. This is from an endometrial biopsy and again with the finding of nerve cells in the endometrium of one with endometriosis, I believe this very well may be arising from the stem cells that are there within the uterus but interestingly, when we looked at these neurons and you can see they sort of form these axons and they connect to their surrounding cells, but when we look to see what type of neurons we can generate, at least one subset that we could generate expressed an enzyme called tyrosine hydroxylase, which is the rate-limiting enzyme in dopamine production. Now, dopamine loss is what is associated with Parkinson’s disease. We show that these cells in culture could actually make dopamine and then we looked at the electrophysiology and show they have the electrical properties and that they would respond appropriately as dopamine-producing neurons normally would in the brain, and we tried them in a Parkinson’s disease mouse model. We treated these mice with an agent that would destroy their dopamine-producing cells in their brain and then we did a transplant of the stem cells from the uterus and here we took the mice, we treated them with MPTP, which causes Parkinson’s disease. We then either gave them an injection of our, what we call, human endometrial derived stem cells or with just saline and we looked here at the markers, this is nestin, which is a human nestin, which is again human cells injected into a mouse brain. So, these were cells that we injected in from the stem cells. They localized to the area where you would normally expect Parkinson’s neurons, these dopamine-producing neurons to be located. We could show by finding human DNA in the brain that these cells, human endometrial stem cells engrafted the brain. They migrated to the area of the lesion and localized there and finally what we did to see if this had real benefits to the mice is after we allowed them to engraft, we looked at the dopamine production in the brains of these mice. Here are our normal mice. They have lots of dopamine that they make. Here are our mice that we treated with MPTP causing Parkinson’s disease in the mice and you see they have much lower levels of dopamine production and when we transplanted them with the stem cells, the dopamine levels went up significantly. So, it suggests that these endometrial stem cells may not only have a downside causing endometriosis but perhaps potentially have some real benefit that we can use them for regenerative medicine and perhaps junctionally even helping to treat people with Parkinson’s disease.
So, I will conclude by saying that that there is a tremendous cell trafficking going on in the body that we are only beginning to understand. Cells move in and out of the uterus, probably necessary to regenerate the endometrial lining with its dramatic tremendous turnover that it has in each menstrual cycle. These cells can come from areas outside of the uterus, and I think that makes sense. We see the women who have, say for example, Asherman’s syndrome are more often pretty good at opening up their cavity and seeing an endometrium regenerating. We are not so good at destroying endometrium. If we ablate an endometrium or we resect it surgically, even if we surgically remove every endometrial cell, these women often still menstruate. So, it may be that even if we do that procedure perfectly and destroy every endometrial cell, cells can come in from outside of the uterus to regenerate that endometrial lining. Stem cells clearly are contributing to endometriosis and again I think they contribute to peritoneal endometriosis to some degree and probably are largely responsible for some of the lesions that we see outside of the peritoneal cavity, and finally the potential is there for us to use these endometrial stem cells for potentially regenerative medicine and other beneficial purposes. We can generate several different cell types from endometrial stem cells. I thank all the people in my lab who have done this and thank again the organizers for inviting me. Thank you.
I have been informed we have five minutes for questions and if there are any questions in the audience, then maybe the speakers can come down or stand up and answer them.
: Do you know if in reproductive age group, there is more prevalence of Parkinson’s disease among the men than among the women?
: Ya, I think the question is getting into do these endometrial stem cells regenerate neurons in women more readily than men? I do not think probably that that is the source of stem cells going to repair brain in normal women. My guess is there are local stem cells there or it maybe bone marrow-derived stem cells that are contributing to that rather than cells migrating from the endometrium, but it is a good question and good thought and I do not know the answer to that.
I think it is very exciting just to wrap up from this morning to here about so many different individual projects when you hear about, endothelial growth factors in the cytokines and the monokines and you know, we have been hearing this for the last 15 to 20 years as far as endometriosis research and now the stem cells, but again we have never been able to put it all together in an entire system and I think that is the exciting work that Dr. Griffith presented this morning. We take each individual in part and each individual _____ and now using mathematical modeling, we will be able to put it together in a system and hopefully come up with some very, very exciting outcomes and data. There are any other questions?
: Yes, I want to ask a question about, I am an example of a woman who has been diagnosed with stage IV endometriosis very late in the game and it was just last year and I am still struggling to find an appropriate physician that will say something beyond you have exhausted your treatment options and even why you wanted hysterectomy, but even going back, I was diagnosed with my first autoimmune disease in 1988, so I was really interested in hearing more about, you know, the links with that and overall for treatment, you know, any of those things. They are very difficult areas and wondering if you have any suggestions?
: Well, it is a good say for the next segment because you are going to be hearing from what I feel are some of the world renowned expert clinicians not just surgeons for the treatment of endometriosis and hopefully will present some new alternatives and options that you have not been presented with.