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SCIENCE AT THE SHINE DOME 2005: ANNUAL SYMPOSIUM Recent advances in stem cell science and therapies 6 May 2005
Kidney regeneration using stem cells – fiction or feasible?
We are going to be changing organ here: as you can see, we are into the kidney. The question is: is regeneration using stem cells fiction or feasible? Obviously, it is fiction at the moment, but I would like to talk through some of the ways we might address this and move it into the more feasible realm.
Here we have the problem: chronic renal failure. Most of you would know where your kidneys are and pretty much what they produce in terms of urine. You might not know that your kidneys also regulate your bone density, the number of red blood cells you have, your blood volume and your blood pressure. Chronic renal failure is a very big problem in Australia. It costs about $1 billion, there are about 50,000 patients with chronic renal failure and about 4000 new patients a year. In the US, this is a $25 billion problem – the prevalence of chronic renal failure in the US is shown up high on this chart at right-hand side of the slide. We are about in the middle, about the same as Canada. And the really important thing to take away from this is that the rate of chronic renal failure is climbing steadily, somewhere between 6 and 8 per cent per annum. This is largely because we are increasing the rate of type 2 diabetes. We are all getting type 2 diabetes, and one of the consequences of that is chronic renal disease.
Currently, if you have chronic renal disease and it reaches a point that your renal function is so low that it is incompatible with life – end-stage renal disease – you have two options: dialysis, which could be haemodialysis or peritoneal dialysis, or transplantation. For a person on dialysis, this costs about $50,000 a year and has a very high mortality rate. It only provides about 10 or 15 per cent of filtration rate and so you have to use pharmaceuticals to provide all of the other things the kidney normally does. This is a very difficult life for patients to live with. It is much preferable to have a transplant, but only one in four patients will receive a transplant. In Australia, about one person a week dies waiting for a transplant. We have about the worst rate of organ donation in the world.
So what else could we do about this? Could we regrow a kidney? Could you repair a kidney? Or what about stem cells?
This is a question that we set about answering, but I would like to start off by explaining to you how you got your kidney in the first place. To get a kidney, you actually have to have two cell types: the epithelial cells of a structure called the ureteric bud, and the mesenchymal cells of the metanephric mesenchyme. Both of these tissues come from the same part of the embryo, the intermediate mesoderm. The bud has a discussion with the mesenchyme. The mesenchyme sends out signals to bring the bud towards it, and the bud sends out signals to make the mesenchyme, near the tip, form a ball, an aggregate. That becomes hollow and then elongates out into a long tube, one end of which will link up with the ureteric bud, the other end of which will be vascularised and so your blood then comes in, is filtered, and filtrate which is going to form the urine goes down and out the ureteric bud. That is the formation of one functional nephron, and each of you has somewhere between 200,000 and 2,000,000 nephrons in each of your kidneys. I will show you that again: the ureteric bud comes in, induces a nephron, it branches, and signals from the mesenchyme back to the bud make it branch and then branch again as it grows through the mesenchyme to form your kidney. So it starts out as two cell types, a ureteric bud cell and a mesenchymal cell, and it ends up making about 25 different, specific cell types, all in the right place doing different things.
Our hypothesis was that it may be possible to use stem cells to differentiate interrenal lineages to repair or regenerate damaged kidneys. Now, this might be a renal stem cell, an embryonic stem cell, or an adult stem cell from somewhere other than the kidney, but to really work out whether we could do this we needed an increased understanding of this whole process that I have just described to you. We needed an increased understanding of how you made a kidney in the first place.
So the first thing we did was to try to actually do that at a molecular level. Day 10.5 in the development of the mouse through to adult is when the mesenchyme and the ureteric bud first appear. So this is the very first time that you get anything that is going to form your kidney. We have actually used microarray chips, which will give you the expression pattern of about 20,000 genes at a time, to ‘walk through’ the whole process of kidney development in the mouse and ask what genes are on, at what time.
And so we have a profile, from very beginning to end, of all of these genes during kidney development and we can identify about 3500 genes that change in their level of expression across time. But that is a very complex picture: when we start, we have got two cell types, and when we finish, we have about 25.
So it is really important to do this not just across time but also across space. We have taken the kidney and pulled it apart, using laser capture, microdissection, flow cell sorting, sieving, and actually getting down there and manually chopping the kidney apart, into bits. We have then profiled all of these bits.
We use a number of mouse tools to do this. For example, you see here a transgenic mouse in which all of the ureteric bud and its derivatives are green so we can FACS separate that away from the mesenchyme. We have here another one in which the interstitial monocytes are green, and so we can profile them separately. Here also is some work done by Gina Caruana – who is actually in this room now – where we have actually just cut out the ureteric bud, just after it has done its first branching, and removed the mesenchyme, and chopped off the tips so that we can look at what the tips are making versus the trunk versus the mesenchyme.
This has all generated a huge amount of data. Not only have we accumulated data across time and in specific subcompartments of normal kidney development, we are actually profiling renal disease – acute experimental models, where there is a certain amount of endogenous repair of tubular damage, and also chronic genetic models, which are not reversible. We are asking the question: are any of the genes that give us a kidney in the first place turning on again in the process of these types of acute models where there is some regeneration, and can we mine all this data? We compare it with known stem cell population genes and with information about what is on in other tissues in the embryo. This gives us a very powerful tool set.
You end up with a huge amount of data, though, and what are you going to do with it? What genes do we really want? We actually want secreted proteins, so that we can see if those secreted factors can be useful in repair or regeneration, or we want cell surface proteins, so that we can pull out specific potential stem cell populations and do something with them. So we do our profiling, and we use bioinformatic predictions to say which of the genes that change on the chip are secreted proteins and which of them are encoding cell surface proteins.
We check which subcompartment of the developing kidney the gene has been made in, and then if we are interested in cell surface marker we use that marker to pull out a subpopulation; if we are interested in a secreted protein we can do an assay called kidney explant culture. You can actually cut out a mouse kidney at about day 11, when we have just got a bud and a mesenchyme, and you can grow it for about six days in culture, during which and it will go through the early stages of kidney development – ureteric bud branching, and new nephrons forming (looking here like little red apples). And so you have got a quick assay for factors, and we can ask the question: what are those factors doing to normal development? Even my children can tell me which of the assays shown here are abnormal: the ones shown at the bottom are not making ‘apples’. Their mesenchyme is quite undifferentiated. So that is our first-pass screen for factors.
I have tried to lay the background in terms of the data that we have been collecting. But what are we going to do with it, in terms of renal repair? We actually have about six different programs that we are working through, to try and look at ways of regenerating or repairing kidneys. I won’t have time to talk about them all. As Bob Graham introduced, we are interested in the process of de-differentiation, which is how the newt deals with damage, and we are also interested in bone marrow recruitment, but I won’t talk about that today.
I would like to walk through a couple of these possibilities, though. The first is a simple one: get the kidney to repair or regenerate in situ. We are hoping that we can identify a factor that could be delivered to the patient to simply kick off its own stem cell population or do some sort of repair via transdifferentiation.
To go back to the profiling that we have done, here is an example where we have profiled a model in which we obstruct the ureter and the urine backs up into the kidney and causes damage. Then when you release the obstruction, after 10 days, it goes through a certain amount of regeneration. So we can look for genes that go up during the regeneration phase and compare that with whether they are dynamic during normal kidney development, whether they are expressed by the mesenchymal subcompartment, and then which of them are secreted factors. And then we test them. You see here a factor that we have identified that essentially doubles nephrogenesis in culture, and we are very interested in its ability to drive kidney development and whether that can also be translated to an ability to improve repair in situ.
We have reached the point of administering this factor back into models of acute toxic renal damage, and we are looking at the histology of the kidney with and without the factor, and also the renal physiology. This is really with the assistance of Warwick Anderson’s laboratory, who can manage to catheterise mice – which is no mean feat.
The next one I would like to talk about is isolation of autologous stem cells for their expansion and redelivery to the kidney. Is there a renal stem cell? Is there a stem cell postnatally that hangs around in the kidney and could do something? This would, obviously, be great.
We are looking at these in a number of ways. First of all, we are looking for renal progenitors, based on their expression in the developing kidney. So if we understand what the metanephric mesenchyme – which gives rise to all of the nephrons – makes, we can then go and look for a cell that is similar to it in the adult.
We did this by another profiling approach, where we took the very, very earliest time point when a mesenchyme has decided it will become kidney and not something adjacent to it, like the gonad, and compared it with a region which will go on and form something else.
We have identified cell surface markers that are on in this mesenchyme very early and that might be useful for identifying a progenitor. We think the markers CD24 and cadherin 11 – in combination and not separately, because they go on to become different things – might be useful for finding an adult stem cell.
We are also looking for long-term label-retaining cells.
It has been mentioned before that in the adult the stem cell compartment should cycle fairly slowly. So one way of identifying it that has been used quite extensively is to use a label such as bromodeoxyuridine, which gets taken up by the DNA. On the hypothesis that the cell that cycles most slowly is likely to be a stem cell, you actually give a short pulse followed by a long chase period. We have done this in the postnatal kidney and chased out for, now, 22 weeks to try and find which are the cells that remain labelled.
As you can see here, we see an occasional, often a doublet, in proximal tubules themselves. And we are investigating whether this particular known stem cell antigen is actually marking the same population. We hope to pull them out and characterise them. What we think they would be is a multipotent epithelial progenitor within the kidney, which could be very useful.
We are also looking for proposed stem cell populations.
Bob Graham introduced the concept of the ‘side population’. As he pointed out, this is originally a term used in hematopoiesis to describe the hemopoietic stem cell. You can purify them by flow sorting, based on the fact that they efflux Hoechst dye very fast. It has now been shown that quite a lot of solid tissues have a side population, and we have looked at the side population in the kidney.
Here is a side population in the bone marrow. It represents about 0.1 per cent of the bone marrow. In the embryonic kidney we have a side population of about the same size, and the same in the adult kidney. So it persists out into the adult kidney. We have used expression profiling again to find out what genes this side population makes.
We have actually gone back then and asked: where is the side population? This is ABCG2, which is the transporter that pumps out the Hoechst, and here you can see a number of other novel genes that mark the side population in similar locations in isolated proximal tubular cells. (These are adult kidneys.)
We are interested in whether these cells can actually do anything. The fact that they efflux a dye doesn’t prove they are a stem cell at all. So we have been taking a very early embryonic kidney and microinjecting isolated side population cells from the adult back into it, by soaking the cells in a label called DiI and then asking where they are ending up in the developing kidney.
Here you can see some DiI labelled side population cells. The green marks the developing nephrons in the kidney, and you can also see double positive cells, suggesting that the side population can go in and incorporate into the nephrons of a developing kidney.
But this is very tricky to quantitate. We have attempted to see whether a side population introduced into kidneys in this way is any better than main population – that is, the rest of the FACS plot that you saw. Here you can see the data for main population and the data for side population, and in only about 1 per cent of cases does main population end up becoming ureteric bud cell, and in only about 9 per cent of cases is metanephric mesenchyme its final fate. We haven’t proven yet that they are actually making the right proteins, but that is where they are located – in contrast to the side population itself: on about 30 per cent of occasions they turn into what appears to be metanephric mesenchyme. So there is considerable enrichment of side population’s capacity to position itself in the right part of the kidney.
We now are moving on to the phase of actually taking side population cells and, again, putting them into a mouse model of renal disease – in this case again it is a toxicity model, but we are looking at a number of other models – and asking what that does to the histology or pathology, the physiology. We can trace the incoming cells because in this case they are tagged with green fluorescent protein.
So the next possibility is using embryonic stem cells. You could evoke this for any other non-autologous stem cell, but we are working on both mouse and human embryonic stem cells. The idea here would be to take your pluripotent cell, convince it to become a renal progenitor, isolate it to purity, expand it and deliver it back to the patient.
Mouse embryonic stem cells have been shown in culture already to be able to be turned into blood, and this is an example of that. Shown here is time against the expression of a variety of genes. You can see that under certain conditions you can get a mouse embryonic stem cell colony to start turning on brachyury, which is a gene saying, ‘I think I am mesoderm,’ then FLC1, TAL1, GATA1 and eventually haemoglobin itself. So you can get this induced differentiation of mouse ES cells, and what we are trying to define, using our expression profiling, is what we would want to see if we were convincing it to become kidney rather than blood.
How are we going about convincing it to do that? We are using cultures of embryonic bodies, which is little clumps of mouse embryonic stem cells, in different growth factors and then testing for what turns on, and we are also co-culturing clusters of green-labelled embryonic stem cells with kidney cell lines. For example, you see here a cell line from the collecting duct of the kidney. I would just like you to look at the panel at the top, third from the left: the DAPI shows you that the clump of cells here has nuclei, the green shows you that they are ES cells, the WT1 marker indicates that these cells, which previously didn’t make this protein, are now turning it on. So we are seeing here the induction of a renal marker. We then take the cell lines that we find are doing this, go back and profile them, and ask what secreted proteins those cell lines are making, that others are not.
This is also work that is going on in Martin Pera’s lab, using human ES cells. (That, I would say, is probably less advanced, because we know less about how to control human ES cells than we do about mouse, which has a much longer history.) Andrew Laslett, in Martin’s laboratory, is now starting to introduce differentiated human embryonic stem cells into mouse models and using GFP to trace what they do and what they turn into.
The final possibility that I want to talk about is really the most ‘out there’ and, some would say, ambitious, crazy. It is to actually bioengineer an entire replacement organ. The fact is that when someone is diagnosed as having chronic renal disease, the clinician will wait until they are, essentially, at the point of no renal function, or insufficient renal function to survive, before they do any sort of intervention. So a lot of these patients are not detected early, so by the time you went in to do some sort of cellular therapy the damage would be too profound. The question, then, is whether we might have to actually make some sort of replacement tissue. There are a number of things we would need for that. We would need some source of cells, we would need the appropriate factors, and we would need some biomatrix or scaffold in which to do it. Then you would have to put this into the peritoneal cavity and get the recipient to vascularise it, and start to get nephrons forming. And then you would actually have to plumb it, because if you get it to filter, it has to have somewhere for the filtrate to go. The obvious thing to do is to plumb it back into the bladder, because that is how urine normally gets out.
In preparation for this – obviously, we don’t have a stem cell source that we can do this with yet – we are investigating an approach where you can take an embryonic kidney, move it into the abdominal cavity of an adult mouse and just put it on the body wall. Within a week it will vascularise, it will proceed in development and start forming nephrons that start creating urinary filtrate through them. So we are now pushing this back to see how early you can go: how simple can it be when you put it in, for it to remain aware that it needs to form a kidney and know how to do it?
As I said, the real problem here is getting that plumbed. Julie Campbell is working with us on that issue of tissue engineering a replacement ureter. The way that is done is to place a scaffold in the shape of a ureter into the abdominal cavity, and have cells from the peritoneal cavity coat that; then, basically, insert that artificial ureter, which ends up getting coated by urothelium – which is what it should have ultimately. And that would be the outlet for the urine from this bioengineered kidney.
So that is a very quick trip through four of the six possibilities that we are investigating, and pretty much where we are at the moment. I think it is still very much fiction. We haven’t been able to pull out a side population cell or a potential renal stem cell and really prove that it is self-renewing and exactly what potential it has. We haven’t been able to investigate yet whether side population is acting as a niche or is actually transdifferentiating, or is simply fusing. All of that is really in the future.
I would like to thank a lot of people. Shown here is the Renal Regeneration Consortium.
We are geographically separated. A portion of this group is at the University of Queensland, in Brisbane – the chief scientists involved in this group are myself; Sean Grimmond, who has really been instrumental on a lot of the expression profiling; Rohan Teasdale, who has done the bioinformatics; David Hume, who is involved in the bone marrow studies (which I haven’t talked about); Andrew Perkins, who is working on mouse ES cells; Julie and Gordon Campbell, who work with us on the artificial ureter. And the other arm of the consortium is based at Monash University – John Bertram works on kidney development, as I do; Warwick Anderson, on renal physiology; Gina Caruana, who is a development biologist as well, is working with John Bertram; and Sharon Ricardo has set up most of the mouse models. Last but not least, we have Martin Pera, who does all the human ES cell work. I would really like to point out a number of people in particular. Stephen Bruce is a PhD student who has been working on the mouse ES cell work; Andrew Laslett, on the human ES cell work; Anita Cochrane is a PhD student who has done all of the profiling of the renal disease models; Brooke Gardiner did a huge amount of expression profiling; Darryn Taylor deals with all of our data. And I would like to point out Grant Challen, who has got to be one of the most dedicated PhD students I have ever had. He took on the Herculean task of isolating enough embryonic and adult side population from the kidney to actually expression-profile it. It is the first time it has ever been done for a side population in a solid organ.
Questions/discussion Question – I wonder if you could comment on this, Melissa. It is not surprising that you can put an embryonic mouse kidney into an adult mouse and get it to grow, because kidney development in the mouse mostly takes place after birth. But in the human it takes place predominantly in the fetus. Do you think that it is going to be more complicated for the human kidney? Melissa Little – I don’t think we are proposing that you would harvest bits of human kidney and get it in there, growing a bioengineered kidney. We are just using this as a model to understand how simple a cell can be, and what it looks like when it is that simple, for it to go through this process so that we could get a stem cell to that state – what sort of renal progenitor would we want to use? Question – I was thinking slightly differently from the other questioner. If you are taking the kidney out of the embryo, have you thought to see whether a second one can regenerate in the embryo? Melissa Little – Yes. It doesn’t happen. If that mesenchyme is gone, you simply have renal agenesis on that side. Question (continued) – Even if you took out, say, 90 per cent? You have got a huge number of stem cells still there, right? Melissa Little – But you have taken out the spatial context, I think, to make it from scratch again. What happens, if you are missing one on one side – you can do this experimentally at various stages – is that the one on the contralateral side gets bigger and compensates. There are probably a couple of people in this room that only have one kidney, and don’t know about it. It is because one of them failed during development; the other one gets bigger as a consequence. You can cope pretty well with even considerable kidney resection; you will still have enough filtration to be okay. Question – This is a comparative anatomist’s comment. The large whales have many kidneys, all about the size of a human kidney or a bit bigger, and that is determined by the limitations of the length of a nephron that can be functional. I don’t know whether they grow all their kidneys when they are in utero or whether they add kidneys as they grow larger, but it might be an interesting model for what you are trying to do. Melissa Little – Oh, I don’t know how I would keep a colony of whales! I love hearing this comparative stuff. I didn’t know whales had multiple kidneys. In fact, all of us have had three sets of kidneys at some point in time. We go through a number of sets of kidneys during development. I think that is interesting, because I really think with these bioengineered kidneys we are ultimately going to have to have multiple ones to do the job. I don’t think you could really recapitulate well what we have managed to do in our own metanephron, or our permanent kidneys. But there is another piece of comparative biology that you may not know. That is, while newts don’t do it, in terms of regenerating organs, you can chop out a stingray’s kidney and it will regenerate. It is the same with certain sharks. And it would be very interesting to understand why that is the case. Question (continued) – But they are using the mesonephric kidney, not the metanephric kidney. Melissa Little – That’s true.
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