At the British Neuroscience Association (BNA)’s Festival of Neuroscience in April 2019, we were lucky enough to sit down with some influential neuroscientists to discuss their work. We’ve assembled these transcripts into our BNA Interview Series. Here, we interview Lund University’s Professor Anders Björklund on his foundational research into the use of stem cell transplantation therapy for Parkinson’s disease.
Ruairi Mackenzie (RM): Neuroscience has recently begun to appreciate that the brain is not a fixed organ – it changes and regenerates over time. How have we been able to show this?
Anders Bjorklund (AB): Well, of course, it’s true that the adult brain and spinal cord doesn’t regenerate very well or in many instances, not at all. That is, of course, a challenge to modern neuroscience research in the first place, to try to understand why is it so? There is one simple explanation, saying that the brain is too complicated to allow feeble efforts of regeneration because things may just be screwed up [during the regeneration]. If that’s the case, then the dampening or blockade of regenerative efforts from neurones in the brain would be a rational approach from the point of view of the brain itself.
Now, we have to somehow, prove that this is wrong. Our main interest is to help the brain to repair itself. Ideally, one would like to see the brain being able to do the same kind of healing as, for example, the skin. If we get a skin wound, the tissue orchestrates a regrowth and we will get something back that is very close to normal skin.
It should be possible, and the idea is that one should be able to rejuvenate the developmental programmes that are active in structuring the brain in the first place during development. It is true that research has been able to show that many of these mechanisms that govern regeneration and govern development are also present in the adult brain. For example, long distance axonal projections can be reconstructed by transplanting cells. If you introduce new immature cells into the brain or spinal cord, they will be able to elongate and grow along the appropriate trajectories and also, find the right targets and hook up.
Clearly, the guidance mechanisms and target recognition mechanisms that are underlying correct connectivity, they are present in the brain but not actively utilised in the scenario of a trauma for example. There is much greater hope today, I think, that we will be able to control and also, make use of the intrinsic regenerative properties that help the brain to repair itself.
RM: In modern neuroscience, stem cells are a useful and relatively common part of our arsenal of neuroscience techniques. When you started investigating the brain, were stem cells or neuroblasts being used in this way?
AB: Well, of course, when we started in the 1970s. The situation was quite negative because early attempts to introduce new cell elements in the brain had given very disappointing results. Neurons were shown not to survive. The reason why we got interested in the performance of neuroblasts in the brain came from or was inspired by studies using similar transplants into the anterior eye chamber. This is a classic technique where one can, so to speak, culture cells and pieces of tissue on the surface of the iris. It becomes a little like a culturing chamber with the particular advantage, you can watch it through the cornea. Through a simple microscope, you can actually look at the tissue piece. The iris is very well vascularised, so various pieces of tissue can then be supported by new vascular supply. This had been used for studying interaction of various kinds of tissue pieces that you put in.
At the time when we started our work, there were studies published showing that pieces of foetal brain tissue, if it was taken at a specific stage of development, could be made to survive in the anterior eye chamber. When they then took the iris out, they could show that axons grew out over the iris surface in a very active and structured way. This intrigued me. I thought that if it was possible to get that kind of cell to survive in the eye, why shouldn’t it be possible to survive in the brain? The first approaches were more curiosity driven, to see what can we achieve with this? Additionally, we had this new histofluorescence technique that was developed by my supervisor in the lab in Lund. That allowed very selective visualisation of neurons using dopamine or serotonin as transmitters.
This allowed, for the first time, to visualise specific neuronal population in its entirety. The cell bodies, the axons and terminals. In contrast to what had been possible to do with standard techniques like silver techniques, that were commonly used at the time, we could trace with high precision, the axonal growth from the graft itself. That became the important trick that we used. We took foetal neuroblasts of dopamine neurons and serotonin neurons and transplanted them. Initially, into the hippocampus because it has a nice structured anatomy and we could show the ability of these neurons to survive, to grow, to establish innervation patterns that were virtually identical to the normal innervation patterns. This then inspired us to move onto the Parkinson’s model that had just been introduced using the 6-hydroxydopamine toxin, which was a new thing. You can inject it into the substantia nigra and wipe the dopamine system out.
RM: And that would reconstitute the effects of what might happen in Parkinson’s disease?
AB: Yes. You get a motor impairment that is quite nicely measurable, but the other thing is that when you inject this toxin on one side, you can wipe out the dopamine innovation in the same hemisphere, whereas the other hemisphere is left normal or intact. We used that model then. That was, I would say, the breakthrough for us, which we did in 1979. It’s actually 40 years ago this year.
RM: What’s involved in the transplantation process in a human brain – are the cells injected? How are they introduced into the brain?
AB: They are. Our initial approach was actually to take small pieces and put them in but that makes it difficult to reach any point in the brain. In the second step, we turned it into what we call a cell suspension technique, which is essentially what scientists use to generate dispersed cell cultures. We adopted that technique with which we could inject defined volumes of cells into, in this case, the striatum. By carefully selecting the age of the donor cells, they can survive sufficiently well. Then, in the next step, we got the permission to do similar experiments with tissue from aborted human foetuses. I think for us, the situation was favourable in the sense that in Sweden, abortion is generally admitted and not a very controversial procedure.
We got the permission, initially, to do this in rats under immune suppression and we could show that we could get the same type of preparations from human foetal material, transplant to a rat model and get them to survive and function. In the next step, together with my close collaborator, the neurologist Olle Lindvall, we then approached the ethical committees to ask whether we could be allowed to do this in patients. That turned out to be a major process for us, where the Swedish Society for Medicine stepped in and their ethical delegation worked out guidelines that then were built into the Swedish transplantation law.
It allowed us to move on and do the first patients and the procedure then is that we found out that one had to collect cells from four to five foetal donors in order to get sufficient cells for transplantation on one side of the brain, which means that for a single patient, we would need tissue from eight to ten donors where the surgery was made in two stages. First one side of the brain and then, later on, second side of the brain.
RM: What did you see happen in your patients when you transplanted the neurons? What was the clinical effect?
AB: Well, the first two patients showed no effect. What was important at that point, was that positron-emission tomography (PET) had just been introduced for studying the dopamine system in the brain. This is a radioactive imaging technique and the main approach for its use in Parkinson’s is to use a tracer for fluorodopa. Fluoro-18-DOPA is a tracer that can be imaged with the PET technique. That had been introduced just a couple of years ago and it allowed us to show that in the first two patients, the cell material had not survived. There was no fluorodopa signal growing up around it. We found out that the technique wasn’t good enough and after refining it, in the next round of patients, we got the expected survival. We could monitor the growth of the transplants using the fluorodopa imaging technique and see how the signal was growing as the cells grew axons into the denervated putamen. Patient numbers three and four, showed very clear and very encouraging recovery also, of their motor function. That made it profitable for us to continue, so in the Lund programme, we made a total of 18 patients over the following ten years.
RM: How many was the treatment successful for?
AB: Well, I think around a third showed therapeutically meaningful improvement and we’ve also been able to follow some of them over a long time, up to over 20 years. There are now several cases where the improvement had been sustained over a long time. We have also, within the last few years, been able to study their brains after they have died, simply of natural causes and been able to see that the transplants show very good long-term survival and most importantly, the reinnervation of the putamen is substantial in those cases.
RM: Why do you think it works in some people’s brains and not in others? Is it something to do with the transplantation or is it something in the neural environment?
AB: It’s something we are debating, of course. I think one should be clear about the fact that this foetal cell material is impossible to standardise. It will vary, depending on how it’s procured and what kind of contaminations there may be. The actual dissections, the pieces that are being taken, the composition of the cell material, the viability of the material depending on how long it’s been around. All this together, means that we had to accept that each cell implant is different from patient to patient. Now, it doesn’t preclude that there is an important aspect of patient selection also because it’s clear that the dopamine cell replacement is meaningful only in patients where the major motor impairment symptoms are due to loss of dopamine. In other words, patients have to be responding to L-DOPA therapy [which most Parkinson’s patients are given] in a good way.
It means that patients shouldn’t be too advanced because over time, many Parkinson’s patients will develop more and more non-motor symptoms and that reflects the fact that the disease process is expanding beyond the dopamine systems. In the initial period, the first five to ten years of the disease, the major cause of disability is the loss of dopamine neurons but the disease process, which is due to protein accumulation and aggregation, will progress. It’s important to choose patients that are relatively early in the disease.
RM: In the patients you followed for decades and in whom the cells were surviving, did they exhibit these non-motor symptoms or did stopping the motor symptoms stop the progression into other areas of the brain?
AB: I think the indication is that the underlying disease is unmodified. In the good cases, the motor symptoms can more or less disappear, and it allows them to stop dopamine medication, but it doesn’t interfere with the underlying progression. For example, we’ve had Parkinson’s patients that had developed dementia over time despite the functioning dopamine transplants.
RM: We now have induced pluripotent stem cells (iPSC) and you’ve mentioned customised stem cell uses now. Are they improving outcomes for patients?
AB: Well, I think it will be a very important step forward to be able to use fully standardised cell material. One advantage with the stem cell derived neuronal preparations is that they will be uniform. They will be generated in large numbers, then frozen down in aliquots which means that every patient will receive the same cell preparation.
We can also control the composition of the cell material. For example, there are clear indications that contamination with serotonin neurons in our transplants from foetuses may have negative consequences. We know, with the [iPSC] preparations, that they are entirely free of serotonin neurons, which means that another element of variability will be eliminated. Otherwise, I think if we look further down the road, I think the main, the most important impact of dopamine cell replacement in Parkinson’s will be when it can be combined with other disease modalities that are slowing down or blocking the underlying disease process. This could, for example, be a gene therapy-like approach where it may be possible to block or reduce this protein-driven pathology that is most likely an important cause of degeneration. A combination then, of cell and gene therapy where the gene therapy is slowing down, and cell therapy is restoring what’s lost. I think that could be the most powerful application.
RM: Sounds like a really exciting glimpse into the future.
AB: Yes, and that’s what we look forward to.
Professor Anders Björklund was speaking with Ruairi J Mackenzie, Science Writer for Technology Networks. Interviews have been edited for length and clarity.