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What does the future hold? - (full conference transcript)

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Professor Richard Gardner

Richard Gardner obtained a First Class degree in physiology at Cambridge in 1966, followed by a PhD under the supervision of Robert Edwards, who with Patrick Steptoe developed human in vitro fertilisation. In 1978 he became Henry Dale Research Professor of the Royal Society in Oxford University and since 2000 he has held the position of chair of the Royal Society working group on stem cells and therapeutic cloning.

I'm very grateful to Ian and his colleagues for extending this invitation to me, because it is an unfortunate fact that all too rarely do those doing research in biomedical areas actually get the invitation to meet patients and hear far more about their concerns and priorities. My brief, despite the very general title, is actually to consider stem cells and practically to give you some flavour as to reasons why these are felt now to be of great therapeutic potential. I am going to divide my talk into three sections, In the first of which I want to just to introduce you to the scientific developments that led to the notion that stem cells might have great promise for the repair or the replacement of damaged organs and tissues. I then want to go on and look at the options for the sources of such stem cells before finally going on to the more important part and more neglected part and that is some of the barriers to actually to applying the principles of what stem cells offer, to practical problems.

So I want to begin by discussing the threads of the science, and Robert Edwards who was my PhD supervisor in Cambridge and who together with late Patrick Steptoe pioneered the development of Human Invetro Fertilisation. This meant that very early stages of human development were actually available for observation, culture, and for the production of certain cell lines as well as the primary purpose, which was of course a revolutionary treatment to infertility. So their main advance was made in 1969 where they first achieved fertilization outside the human body, and then of course nine years later this led to the birth of Louise Brown and improvements in culture conditions, which meant instead of achieving fertilization and replacing the earlier concepts back into the womb at the earliest possible stage, they could culture on to the so called blastocyst stage that I will mention later.

Listening to Professor Nicholson earlier this morning, my only regret is dear old Patrick Steptoe was not alive today to hear his presentation on laparoscopic, or keyhole surgery. What you may not know is that Patrick Steptoe, who worked at Oldham General Hospital, was the original pioneer in this country of keyhole surgery, and about the time he met Edwards a learned committee of the joint Royal Colleges was deliberating about the keyhole technique. They produced a very fat report saying that these techniques had no future in medicine or surgery. It is a classical case where you would like to meet the committee and make them eat copies of their report in public.

The second scientific thread, which was a very key development that was achieved by a colleague of mine in Cambridge called Martin Evans, was to grow originally in a mouse, to grow the fertilised egg up to this so-called blastocyst stage, and then of replacing it in the womb, convert it into a culture, and what they achieved is that pool of unspecialised cells that is present that persists in the early conceptions. Most of the cells become specialised very early to form structures of the placenta that will attach the future foetus to the mother, but this little pool of cells that remain unspecialised by culturing the blastocyst. Under certain conditions, you can get these cells to carry on growing indefinitely, but without becoming any more specialised. So as it were marking time, they are increasing in number, and can be grown over a long period of time, but they are not becoming anymore specialised. Whereas if they remained in the context of the whole blastocyst, they would carry on with their development.

The third scientific development, which I think because people get too often the notion that scientists are just mindlessly tinkering with nature, I ought to explain a bit, and this is Nuclear Transplantation. This involves taking the genetic information from a body cell and putting it into an egg whose maternal genetic information has been removed. Why was this originally done? Was it to address an absolute fundamental problem of biology? That was to know as cells become specialised, in a muscle cell for example, what happens to all those genes that do not have to be active in order for that cell to be a muscle cell? And indeed if they were active, the cell would be in a totally confused state. So we know in muscle, nerve, skin, intestine, all these different types of cell, different subsets of the 40 or so thousand of genes that we process are actually active, and the question was, what happens to those genes that are not active? We knew all types of cells contain the same amount of DNA, so they presumably retained all these genes, but were they permanently switched off or modified or not? Looking at this problem you cannot think of a more critical test than taking the genetic information from a specialised muscle or nerve cell, putting it back into an egg, and saying: Can that genetic information support the development of a new individual? If it can, it tells you that all the genes are still accessible to being switched on. This was basically the result that was first achieved in frogs, and then in the very famous case of Dolly the Sheep, which was important in showing that a specialised cell from an adult still retains this capacity to be re programmed completely so it can support the whole of embryonic development.

So those are the three scientific threads whose intertwining has brought us to the position where we are now. One of the things that I find unaccountable in this story is the fact it took our scientific colleagues so long to cotton on to the idea of tapping the potential of embryonic stem cells, And I again return to this man pictured Bob Edwards (see photograph, left), when we were celebrating his 75th birthday with a conference in Venice some three years or so ago. It was again this person, who in 1982 wrote some explicit articles saying, given that In vitro fertilisation for treatment of infertility generates large numbers of spare embryos, he specifically looked at the question of the possibility of using spare material for therapeutic purposes.

At that time he considered two options, The first was to grow the embryos on to a slightly later stage to the time when they form the rudiments of basic organs and possibly use those for transplantation, and that was an option that was closed by the Human Fertilisation Embryology Act, which limited the period you could achieve in vitro development to 14 days. That is up to the stage of the threshold of the beginning where the future foetus starts organising itself. But he did also look at the other possibility based on Martin Evans work next door of getting embryonic stem cells, and an alternative would be if you could culture these spare human embryos through the blastocyst stage, could we then get cultures of human embryonic stem cells that would grow indefinitely, but we could keep in an unspecialised state for as long as we wished?

It was unaccountably almost 15 years before Edward's explicit statement of this was adopted. So we go from 1982 to 1998 when Thompson in Madison produced the first Human Embryonic Stem Cell Lines, and it is very curious to me why it took so much time to adopt. So I now want to ask a little bit about embryonic stem cells, why are they so special? Why are they so interesting?

For ethical reasons the sort of studies that really define these cells cannot be done in the human, so we basically work in this case in the mouse and what you see held here on a pipette (see photgraph to the right) this rounded structure is the blastocyst - the human one is very similar to the mouse except it's slightly larger. It's like a sort of outer ball of cells - these are the cells that will form the basis of the placenta. Stuck against the inner surface in a disc is the so-called inner cell mass which will form other membranes but contains this little reserve of cells that are completely unspecialised which you can then extract and culture to get embryonic stem cells.

The critical test that demonstrates the great potential of these cells is that we can take a single embryonic stem cell from one genetic strain and we can put it into a blastocyst of another genetic strain and because the two are different we can actually see exactly what that cells do in the resulting mouse. It's a coloured blue cell and you're putting it into a red embryo and you can regard it in that way with a genetic marker so you can see exactly what it does later on in development. The answer was very striking because this shows you a mouse foetus all in red (not shown here) is what's formed by the progeny of one cell. In other words that all two hundred or so cell types of an adult can be derived from this one cell.

That's an absolutely crucial test that tells us embryonic stem cells, if we can find the right condition, can form every type of cell of the adult body. But life is never simple and straightforward, and no sooner had that bio medical community cottoned on to the possible benefits of embryonic stem cells than people started to find that cells from adults were far more versatile then we'd assumed hitherto. I just list here (diagram not shown) some of the conversions of cell types that have been observed and some under very interesting conditions, notably where women have been recipients of bone marrow grafts from men. So you can actually use a special probe that identifies the wide chromosome that's characteristic of men to see where these cells go and the assumption was that bone marrow cells would just re-populate the entire blood system, but they do more than that and confine male cells are forming card carrying liver cells, there colonising the kidney and various other organs albeit at relatively low level.

So this raised a question of whether you needed to use embryonic stem cells because many people, for ethical reasons, are deeply concerned about the use of these very early stages of development for such purposes. At the moment it's really too early to say how valuable adult stem cells will prove because a lot of the information is anecdotal and unfortunately some, particularly those in the pro life movement, have really rather grotesquely exaggerated what's been achieved with them so far.

But there is a third source of stem cells which is very interesting, and these are stem cells, which are found in the placenta and the umbilical cord when it's discarded at birth. These stem cells are quite rare, they have been used as an alternative to bone marrow for restoring the blood system after damage, but people have not yet succeeded in growing these cells in culture before they freeze them down, so it's a matter of how we can overcome this problem and amplify them and then investigate the extent to which they will convert from one cell type to another. The wonderful thing about those, if you could really harness them of course, is that it's an unlimited supply of material, it doesn't raise great ethical issues because it's in an organ and tissue that's actually normally discarded at birth.

If we actually look at this question of embryonic versus adult (see Table above), in a dispassionate way, you can say that both of them have positive features and drawbacks. As I have mentioned with the embryonic stem cells, we know they're able to form every specialised type of cell in the body, we can obtain them in large numbers, and they seemingly have unlimited capacity for growth. Some of the human cell lines have now been kept growing continuously for three or four years without any sign of genetic deterioration. The drawback is the only source is the very early embryo, and they don't offer an immediate prospect of histology compatible or tissue matching to a patient at this stage. With the adults stem cells we avoid the use of early embryos, it does offer the prospect of a perfect tissue match, and another important point is that embryonic stem cells in their unspecialised state, are capable of forming very prevalent tumours, so you mustn't have those in a graft. But the problem about adult stem cells, in most cases they are actually rather difficult to obtain, certainly from living individuals, their growth potential may be rather restricted, and they may have suffered genetic change during the course of their history of development. So there are two sides to both of these types of cell, but more work certainly needs to be done with the adult stem cell before we know how useful they are.

Now if we come onto the third part, it's really harnessing these cells, saving the life of one individual who requires a graft or a transplant can only be achieved following the death of another individual. There is a permanent shortage of organ donors, and some people become very sick indeed or don't survive to the stage where they could benefit from a transplant.

The idea advocated by the British Medical Research Council some years ago, that we should all be opted in and have to opt out of donation, I think after things like Shipman and Alderhey went down like a lead balloon, so there still is a very serious problem there. And the idea of xeno transplantation i.e. using animals, perhaps genetically engineered so as to be tissue matched to humans, as an alternative possibility, have great set backs as a result of both HIV infection in the human, and of Prion disease like the new variant CJD, which tells us that a micro organism, a virus or a related micro organism, that is adapted to a particular host, after all its not in the interest of a parasite to kill its host, they co-adapt to each other so the parasite takes what it needs without excessively compromising the host, if they jump, or are carried across species, the relationship isn't balanced , and the results can be absolutely devastating.

What people are beginning to look at is an alternative whereby you would have banks of embryonic stem cells that were pronounced virus free, and tissue typed that could be there in anticipation of patient needs. The possibility that you could actually catch the development of disease or damage to an organ, at an early stage, so that seeding it with healthy stem cells might become a viable alternative to actually removing or replacing the entire organ. That's the lines on which people are thinking about in this area, and that addresses one of these main problems, and that is the question of availability. The other problem of compatibility is a big one, but if you imagine, depending on the size of the bank of stem cells that you actually had, the scope for achieving a match.

Talking to my clinical colleagues, with embryonic stem cells if you had banks of two or three thousand or more of these cells that were representative of the genetics of the population, you could already achieve probably a better match than happens with organ transplantation at present, and with the increase in the size of such a bank of cells, you could do progressively better. But it doesn't entirely solve the problem of matching, or avoiding a miss-match, so it's that that has led to this other notion that has the very inappropriate appellation of therapeutic cloning.

Basically this starts in exactly the same way as producing Dolly the sheep. What we do is we take a specialised cell from an adult, we take its nucleus which contains the full genetic information, which is the same for all cells of an adult, and we inject it into an unfertilised egg, whose own chromosomes are completely removed, and then by some electrical, or other stimulus, we start it to begin to develop, but instead of carrying on the development, we wanted to develop to this blastocyst stage, so we can then convert it into a culture of embryonic stem cells. The whole purpose about this, and why its been called therapeutic cloning, is that you have a patient needing a graft, if you take the genetic information from the nucleus of healthy cells of a tissue of that patient, and put it into an egg, you've then produced embryonic stem cells which by definition, are genetically identical to the patient so there can be no problem of graft rejection. But there are formidable obstacles. There are a lot of ethical objections to this procedure, there is already a great shortage of human eggs in fertility treatment, and there are various other reasons produced that suggest, even though this technique works in animal studies, it may not work in the human, because work in the monkey has shown that the eggs react very differently to this treatment than they do in cattle or sheep or pigs.

Among the other questions you have to address is, if you have your embryonic stem cells, we are getting better and better at knowing how, having got the culture growing up, how to push the cells along a particular direction of specialisation. People are now very good at getting the cells to form nerve cells, they're getting better at making the cells form the pancreatic type cells which are responsible for the reduction of insulin and they've had very spectacular results in getting these cells to form heart muscle that will form an integrated sheet of cells that behave in a wholly harmonious way. But the difficulty about this at the moment is, once achieving a bias in differentiation you're not getting a hundred per cent of cells to go in the right direction and therefore if you grafted these cells they would contain an uncertain proportion of cells that were inappropriate for the graft site, and we don't know anything about the consequences of that.

There is the further problem that you don't simply want stem cells in all cases to become specialised, non-dividing terminal cells. If you want to put these cells into a situation in an organ that must continue to grow, you've got to get these very unspecialised stem cells to reach the status of more specialised stem cells appropriate to the organ or tissue in which you find them. Very little progress, so far, has been made in reaching that particular goal. There are various other problems. Essentially what we're talking about with stem cells are cells that are able to produce more of their own type, they're able to renew themselves and become specialised. In a simplest way you can envisage a cell dividing, one of its daughters going on to become specialised, and the other remaining a stem cell. That's what happens on average, in some cases, a stem cell divides and both daughters differentiate, and in other cases neither do, so the population is maintained, but the very important thing I've illustrated here (see Diagram to the left) is that if you want to put stem cells in an organ and get them to carry on growing, they have to be in a very specific niche. We now know their relationship with other cells has to be very specific. If you don't achieve the appropriate niche, then what you're going to have is that both the products of the stem cell division will become specialised, you'll use your stem cells up, and there will be no cells for renewal.

Now when one comes specifically on to the kidney, although great success has now been achieved with the nervous differentiation, and some have you may have heard of recent animal studies that have produced very promising results in dealing with Parkinson disease, and these wonderful sheets of heart cells have been shown to be able to repair damage to the heart muscle after coronary problems, that these are very positive, but when we come onto complex organs that consist of a multiplicity of different cell types, the whole problem becomes much more difficult.

In the case of the kidney, I don't want to preach to those who already know a great deal about it, but the basic unit of the kidney we are concerned with, the basic filtration unit, is the nephron and in mammals like ourselves, the total complement of these filtration units are established round about the time of birth. There's no evidence that any more are generated during teenage or adult life, so we have the basic complements of these units, and as we grow larger, the units grow larger, but they don't increase in number. We know that following chemical damage or various forms of damage to the individual nephrons, if the damage isn't too severe, then cells within these tubules can proliferate and repair damage to individual nephrons. But the whole issue of replacing damaged nephrons is a much more difficult one, though people working on this problem are now beginning to make some progress with understanding the nature of the proteins, the so called growth factors, that are required to actually induce the structures to form and to become specialised. There is some hope in that sense. There is the other problem which is particularly huge in the case of the kidney, and that is the question of whether putting healthy cells into a damaged organ, whether such cells are going to actually survive any better than the cells that are already there. You know as well as I do, if not better, the great problem with chronic renal failure, is how far it tends to progress before it leads to diagnosis. That is another very serious problem.

What options do we have? Well, the options, I think, are much longer term ones, but one possibility to which people have been moving, is actually trying to build an organ in vitro that you might then transplant. The people involved in tissue engineering have made quite intriguing advances with achieving three-dimensional architecture of more complicated bodily structures in culture, by use of various supporting systems. One of the cases was done in cattle, where they took a bull; they took a nucleus from one of his cells, put it into an egg to produce the cloning, and they produced a cloned calf which would therefore would be genetically identical with the original bull, and from that cloned calf they removed cells of the kidney rudiment and they put them on an appropriate polycarbonate scaffold and then they engrafted this back into the adult. Because it was genetically identical, because it was cloned, the graft survived, but it also did show some production of fluid that had the basic composition of urine. This does offer the prospect of taking this further. Whether you would begin with embryonic stem cells and actually get these in vitro to become specialised as the basic cell types of the kidney and then build these scaffolds entirely in vitro, or whether one might take an alternative route which was done in the case of Parkinson's disease, and that was actually to use nerve tissue from aborted foetuses to put into the brain. Aborted foetuses would be another source of the rudiments of kidneys that could act as starting material for trying to build the structures.

In summary, although everyone's getting very excited about stem cells and what they can do, I think for complicated organs like the kidney, the prospects are very much more remote than for relatively simple systems like the heart, or others. I didn't want to come here and be totally pessimistic about this, on the other hand I do feel that within the field, the excitement has generated rather a lot of hype, and in danger of generating unrealistic expectations, and we've been through all this before because about 25 years ago we were told that gene therapy was the greatest thing since processed cheese, and it's only now beginning to yield rather meagre returns.

One final point I'd like to make, which may be something that is further down the line but needs to be explored, and to my view has been neglected so far, and that is that in higher vertebrates like us, we seem to have lost the capacity to regenerate. If a frog finds itself in a difficult situation, having a complete limb severed it can generate a pretty good replacement limb. There is some limited regeneration in humans and other mammals in terms of development of the foetus, but after we are born we seem to have lost that, and I think more work looking into what it is that enables some organisms to regenerate whole limbs and whole organs, whereas we lack that ability, is another area that really needs to have more attention to be paid to it than has been so far.

I thank you for your attention.

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