Cells： from Robert Hooke to Cell Therapy - Professor Anders Lindahl

Well, distinguished guests, colleagues, colleagues at the Royal Society, friends, but also family, please feel wholeheartedly welcome to the second day of the Royal Society discussion meeting. Building on yesterday's sessions on the discovery and biology of the cell and various cell types and technologies, today's focus will be on the delivery of cell therapies into the clinic for the benefit of patients and society. We are truly delighted to have gathered the world's best scientists and leaders in the field of cell biology and cell therapy. And it's a great honor for me to introduce the first speaker, Professor Anders Lindahl from the University of Gothenburg in Sweden. Anders' research on human chondrocyte, and that is cartilage cells, pioneered the use of cell transplantation for the treatment of cartilage defects. Together with Dr. Pettersson and Dr. Britter, more than 1,700 patients have been treated with his cell therapy using cells processed in his laboratory. The title of his talk is Discoveries and Execution of the First Specialized Cell Therapy, Autologous Chondrocytes in Global Use. Please, Anders. And thank you, the Royal Society and Chris, Ian and Johan for inviting me. I will try to give you a 30-year experience in cell therapy based on half an hour. So if I have too many slides, you have to excuse me if I jump a few. I will try to take you through how the concept of cartilage regeneration was seen. It was just taking away the so-called dead tissue. And now we have cartilage regeneration instead. Chondrocytes were considered non-dividing and not possible to do anything good if the tissue was damaged. And the hypothesis of the joint among surgeons was that the cartilage is like the tire of a car. When you wear it out, you just change it into a metal or something. So that was the base when we started. Since we are at the Royal Society, I think this is a very well-cited person, William Hunter, who published this in Philosophical Transactions. If we consult the standard chiropractic writers from Hippocrates down to the present age, we shall find that ulcerated cartilage is universally allowed to be a very troublesome disease. That is, that it admits of cure with more difficulty than it carries bone, and that when destroyed, it is never recovered. So this is the first real thought about cartilage. Why is it untreatable? Well, the patient doesn't come until the patient has problems or you have a radiographic deterioration, and there is no early disease marker. The surgeon treats not the cartilage, they treat pain among patients, and it's a disease of the joint. To the right, you see the destroyed cartilage in osteoarthritis, and on the left, you see the very nice cartilage when it looks normal. This is a huge health problem, and it will increase. I will just point out that in the US, one million patients are hospitalized for operations each year, and the cost is about 60 billion dollars per year. When you talk about osteoarthritis and what we are treating, there are two parts. Primary osteoarthritis, which is actually the long-term disease based on inflammation, and it's the same if you have cardiovascular disease over time, and you have secondary osteoarthritis, which is based on a trauma. These traumatic injuries will lead to osteoarthritis in the knee that is affected, and this is the condition that we are treating with biological regeneration. How common are these injuries in patients, especially in younger patients and active patients? Today, people are more active than ever, and when people go to the surgeon and they do an arthroscopic examination, you have 63% of them have cartilage damage, and about 20% has down-to-bone defects. The question is, why can't cartilage repair? Well, there is an inability of filling the defect. There are few repair-competent cells, and the exposed cartilage inhibits cell adhesion, and the cells are trapped in matrix. This is a textbook from 1937 in histology by Dewar, that the chondrocyte is unable to divide and regenerate itself, and more unlikely repair damaged tissues. When you were a medical student and you read histology, cartilage was doomed. So, if you look over the time, what has happened? Paget presented a paper in 1853 saying, well, the same thing as Hunter, it's nothing to do. Then appears Magnusson from Sweden. He actually did the first successful debridement operation. He took away the damaged cartilage and started bleeding, and the patient became better. He published that in 1941. Of course, the world had other problems at that time, just to talk about cartilage. But his paper was read by Pridy and Johnson and Stedman, that if you start bleeding, you will produce some type of cartilage that might help the patient. Then there's also one by Jackson, where you unload a damaged part of the joint. So, these were the surgical treatment that appeared during the early 50s. You had the first joint replacement in the 1970s with bimetal implants. And then in the 80s and forward, you have sort of a biological approach that was coming up with transplantation of tissues or cells. So, the biological approach was microfracture. You recruit bone marrow trauma cells from the bone marrow into the defect, and then you produce a cartilage tissue. You could also move condocyte pieces, cartilage pieces, by autograft or autograft, the oath procedure. And what you see down here is actually this microfracture where you drill holes or pick holes to start the bleeding. 30% of patients are cured by debridement, but usually the repair is not full cartilage and it fails after three to five years. Who thought about condocytes? Well, we have the pioneering Audrey Smith, who actually cultured and froze condocytes. We have George Bentley, who started the concept of treating cartilage defects in animal studies in the 70s. And then we have from cells to condocytes, and we started, this is just, you have on the left side, you have the condocytes in the cell trapped in their cartilage. It's called lacuna. And on the right, you see when they are left, they are released from Hooke's cells. They are actually out here. And as you can see on the right downhill side, they actually would like to talk to each other and meet each other, as you can see here. So, it's actually, they like to condensate and produce new cartilage. We were the three that were connected in this adventure. I was a PhD student. I'm an MD, but I did a PhD study on the growth plate and growth hormone in endocrinology. And Dr. Pedersen down here, he worked and had done a clinical animal study in the US at Hospital for Joint Diseases with a rabbit. And Matt Sprichberg joined us earlier. We started 30 years ago, so we're looking different at that time. Not necessarily better. There are two animal studies in rabbits that sort of paved the way to the regeneration, and that is actually making a defect in the patella of the rabbit, and then look what it looked after one year. And this was presented at the Swedish Medical Society in 86. And the left side, you see the regenerated cartilage, and the yellow part on the right is the exposed bone. And this is installed from the same. So, we asked ourselves, could this work in humans? And of course, you have to get away from the research environment in order to implant patients. And there was, I would like to recognize Dr. Howard Green in this work, because I did a postdoc at Harvard in his department. And he pioneered cell therapy for burn patients and started biosurface technology. And they supported our work in the early phases. And later, Genzyme bought biosurface technology and the cartilage technology. So, you get things right if people are saying different. So, we combined sterile culture technique and surgical methods. You see on the left side, a small piece of cartilage. We collected the patient's own blood to produce serum, because we did not want to use fetal cap serum. And I think that was a good decision, because we wanted to do as safe as possible for the patient. Because otherwise, we would have actually added the mad cow disease to the patient cultures, which would have been bad. Excuse me if you don't like surgical. But up on the left side, you see destroyed cartilage. It's debrided. And you take a piece of periosteum from the skin bone, and you sew it over and you make a sealed compartment where you inject the And so, this is the publication that we did in New England Journal of Medicine in 1994. It's actually the same concept from the rabbit. And the clinical result is this. The left side, it's a defect down to bone. The right side is a patient going through a second operation on the patella. And you see total regeneration after 46 months. On the left side, you see that it works on the femoral condyle, but not on the patella. And then the patella was the animal studies. There's so much for animal studies compared to humans. How was the response? The media response was very high. It was considered one of the top achievements of that year by Harvard Review. The patient were happy. Scientific community got outraged, because first of all, condocyte were not supposed to be cultured. The osteoarthritis people became upset because something came from Sweden and not from the large societies. And the other thing was also that we were considered unethical. We didn't do the right. We had the wrong methods. We had the wrong animal models. We should have taken care of dogs. We were taken to a meeting with an FDA and a hearing in Washington, and they said, well, you did all things wrong. What's wrong with 50 healthy Swedes? And then the business community came, of course, running after this event. So there are lots of companies starting to see if we can explore this. So between 1994 and 2000, we presented our data and everybody said, well, it's nice, but it's too short follow up time. Okay, so we waited another year and came another year and another year. But then we ended up with five and seven years good results. They stopped the criticism. And we worked on other approaches with tibia, bone and cartilage defects and the ankle. And we also, because of people were starting to do in cultures in the labs and the surgeons came, could you do some cultures for me? I self-inflicted the regulation on myself and got a GMP approval. So this is what it looks today. I will not dwell on all the follow ups that we have done, but two messages. People are getting better after a long time. It takes two years because within two years you have some failures. After two years, if you're good with the treatment, you're good up to 11 years in the study and actually up to 20 years. Although by age you get a little bit worse, but it's a regenerative process where the patients do well. And in most cases, about 70% of the cases where we did biopsies, we cannot do this for ethical reasons anymore. You get high-link cartilage. This is just on the left side you see an MRI with a defect. This is what it looks like for the surgeon. And this is what it looks like after treatment and three years later, almost cured and you don't see any defects. So it regenerates by this. And we are actually seen by MRI and a specific MRI procedure that the 9 to 18 years after ICI, the quality of the repair is similar to the surrounding normal cartilage. So it has a long durability. So what is happening? Well, you see they had the first patient in 88. Today it's over 45,000 patients worldwide by different companies, as you see here. I can't take them all, but I will just, and also people improve the technology. Genzyme worked and put a lot of money into the market. They were bought by Sanofi Avensis and now Oström with very little. I think they put in a hundred million and they were bought now last year for six million. So the business concept of this cell therapy is not sustainable. You have to have something new. Tigenics is approved in Europe and Isotis, I think that's Clemens van Blitterswijk and he produced a cell protect that's actually one hour cell isolation on matrix in the OA for the surgeon. So I think that's a real improvement. So that is getting. We had also Bentis group again, we had collagen membrane instead of the periosteum. And in EU we produced a hyaluronic acid scaffold with condocyte. So you can do an arthroscopic procedure. The first was open. Now we mostly do closed surgery with an arthroscopic procedure where we put this in an implant. Sorry, wrong way. Yes. Based on the first paper from the New England Journal, it's the second most cited paper in the world in the area of orthopedics and the fifth in the world of osteoarthritis and cartilage. So we were happy that it has been accepted. But citations is one thing, reproducing results better for a scientist. So these are actually more, but these are randomized trials out of five. All produce good results. It's a combination of the old technology and matrix. One has a negative result or actually no improvement. I think with our work, we made an impression on the science area. If you look at the number of papers in the, if you look at cartilage regeneration, cartilage stem cells and condocyte transplantation, they have increased tremendously. You have a background increase in science publication by 150%. So what have we achieved in this? Well, I think that we have increased the knowledge. A lot of people are doing research and have followed and improved what we have done. And I just give you a few points on that. These are the different types of cells that people have published that could be potentially used. I must emphasize Ivan Martin in Basel actually, because when they came up with taking nasal cartilage and put that into the knee, if you're a surgeon and you were told when I was a med student, never put your nose into the knee because it's very much for infection. But and I made that joke, but I'm sorry for Ivan. But it actually works. So they've done clinical trials and perhaps due to my comment, I'm not sure, but it's produced a high-end cartilage. So it's another procedure. You don't have to open the knee. So I will work to what we will do further on. Cartilage is an immune privilege. There's no blood supply. So could we derive a universal donor cell and thereby avoiding this single patient culture, very labor intensive treatment. So we worked, and this is a publication together with Johan here, when we started with embryonic stem cells. It's difficult to differentiate embryonic stem cells and chondrocytes, but we did a co-culture with irradiated chondrocytes that were able to, to see up to the right, this is a cartilage noodle. The left side is what is happening if you do this pellet culture with ES cells without the irradiated chondrocytes. I must also acknowledge the work of Susan Kimber and Tim Hardingham, who have done embryonic stem cell protocol, and they actually treated with, in this case, a rat. On the right side, you see that it is an osteochondral defect where they implanted pre-chondrocytes and they produced a nice regenerating tissue. So there is a potential with these cells. And we also, in order to make sure that we can differentiate something better, we used chondrocyte and made an iPS cell line from that based on mRNA. And this is just saying that we had, we checked for the phenotype and the SNPs and the karyotype, and it was all intact after the transplantation. And it seems that the derivation of the chondrocytes, the iPS from chondrocytes, actually produced a little bit better. To the right, you see the iPS cells from chondrocytes. The middle is from a fibromyalgia cell line, and the left is what ordinal chondrocytes do. But there is a problem differentiating embryonic stem cells to ordinal cartilage. So the non-regenerated cartilage, do we have any stem cells there and niches? When people would say, well, it's just de-differentiated chondrocytes and they're useless for your purpose, so we started to demonstrate, couldn't they have more potential if you, mesenchymal stem cell was the one that to be used. But we did parallel work. It was Eva Martin and Friedrich Leuten and Delacchio, who we were able to, almost at the same time, to demonstrate that the chondrocyte had a plasticity. Mesenchymal stem cell differentiated chondrocyte, fat cells and bone, and in vivo they produce bone. And if you do the same with culture chondrocytes, they appear the same, but they produce cartilage in vivo. And you can actually see the same if you take a single cell and clone that. So they have a clone of plasticity in their work. So we could definitely say that the chondrocytes are much more broader in their repertoire than has ever been expected. This is a, when we were looking at stem cells, we actually, this is a work that we published in 1992. We didn't do better pictures at that time, I'm sorry. It's actually a drawing. But this is label retaining cells and we were looking for growth hormone effect. But if you see the shadowed area on the lower right, you see this line and there were cells in the corners of the growth plate. And this is actually the zona ranvier, which is, by microscope, this was discovered and described in the 1830s. But when we did labeling experiments in the same way, we saw that there was a label retaining cell area, probably a thumbtack of stem cell niche, that could potentially support both the growth plate and the cartilage with cells. This was combined with the work together with Helena Brisby, who is an orthopedic spine surgeon. And we injected iron that was taken up by the cells in this niche area. We actually did a fluorescent on the other side just to be sure, but that didn't work. But both on the top you see that the spine and the disc, they also are supported by the same type of niche as in the joint. And the blue dots just indicate where we found cells that had migrated from the niche into the joint. So actually it seems that at least in the growing animal, this is a rabbit, cells are coming from the niche and they are moving into the articular cartilage as well as the ligaments and also the meniscus. And if you exercise a rat, put them running and also drinking BRDU, you actually see that the exercise will release more cells from these niches. So this is something that we are trying to now further understand and if it actually has some influence on the adult individual. So there are two parts I just would like to end and that is osteoarthritis and disease mechanism. What are they? And there are two ways that have gathered data that have changed the view of osteoarthritis. Are stem cells and developmental regulatory genes involved in osteoarthritis? That's the question that we asked and some others asked based on condosites. And genome-wide association studies, there have been several ones and you could expect, for instance, if you take a type 2 collagen mutation, you get osteoarthritis when you are in your teenage. So people were looking for the building blocks, molecular building blocks of cartilage and do we have point mutations or regulatory parts of the polymer that is actually forming the cartilage? But the only thing that is coming up is gene regulation, apoptosis, stem cell control mechanisms and furthermore, the wear and tear hypothesis is now replaced by probably a dysregulation in regulation and low-grade inflammation. I'll just give you a few. This was an array where we compared healthy, so sort of healthy cartilage together with ERSHA-DT in Berlin and we also were able to demonstrate that the notch is expressed on the surface of the cartilage and also we have YAGD, which is one of the connecting signals. But in osteoarthritis, this is highly upregulated, probably a regenerative response to the disease, but it's interesting that we have these findings. So genome-wide association studies and condosite microarray and proteomics suggest regenerative dysfunctions and this is a review by Laughlin who is from Oxford. He is in charge of a large consortium in wide association studies and this is what actually it all comes down to. I think this is a 30,000 to 40,000 patient that has been studied. You have cell proliferation differentiation, condosite development differentiation, hyaluronic acid, which is not strange, transcription regulation, skeletal development, regulation of apoptosis. GDF5, I'll just say one thing about it. There is a polymorphism in GDF5 that is protective in females that has been found for knee osteoarthritis and it's actually a CG domain, so it's actually a methylation site and what the polymorphism is actually that you gain the methylation site because it's actually lost in humans but it's present in all four-legged animals and we looked at 250 patients that we have been operating. I looked at the polymorphism in that site and the interesting thing is there's a majority of men who has been operated but we would have expected 12% which is the polymorphism and out of 150 patients we saw one female who was operated. So it seems that actually females with that polymorphism doesn't go to the doctor after an injury, they regenerate their cartilage by themselves. So the GDF is a central part of this. We will publish this later on. And the last problem with osteoarthritis is that there is no pharmaceutical company, there are no investors who will put one single penny into this disease because FDA has decided that the end point of osteoarthritis is standing x-ray and joint narrowing. The study will take 25 years. Do we have any investors here who are willing to put up the money? No. But there is, on the other side, not a single biological marker that will function as a surrogate marker. So we are trying to address this with, and this is also part of unfortunately too early, we had a collaborative work on fragmentation of the building blocks of cartilage. And we focused on one, it's comp, which is a pentamere that is part of the assembly of collagen. And we raised the hypothesis, what about comp, because you can not on a single individual say that even if you have a cartilage problem you cannot say, we cannot measure the comp in the blood. But are there something with degradation? And perhaps there is a certain time of degradation. So you actually, you build this in a certain way, but you also break them down. For instance, if you are going to take down a house, you don't start working and destroying the basement first. You probably take them from the outside. So we addressed the hypothesis. First of all, we did some proteomics, but we actually turned into the horse. And then people are saying, why are you doing clinical work when you work with the horse? This is the largest horse in the world, this is the smallest, but there are two good things with horses. You can get healthy cartilage whenever you like, because they are slaughtered unless you eat them at the restaurant. So we can make an experimental model where we subject cartilage for inflammation and thereby by proteomics see what is happening by inflammation and the breakdown of the cartilage. So now we have breakdown particles of comp that actually could potentially actually work as drivers of the inflammation. They actually work as what we call metric kinds. This is something that is coming up now that the breakdown of matrix actually cause and continues the low-grade inflammation. And since the domestic horse genome is sequenced, we can just translate that to the human sequence and say, well, these fragments must be in the human also because they have exactly the same fragment. So conclusion, we can say that it's a disease-modifying treatment with long-term clinical improvement. It is not a sustainable business for companies, which means that the people are not getting the treatment that they would. We have been doing now exactly up to 2,700 patients now. We could take everything and we are doing it on a research clinical trial basis. It's adapted to a year regulation of advanced therapies. IPS technology could give future treatments and disease-specific stem cells that can be used for the treatment or embryonic stem cells. Osteoarthritis disease mechanism are revised. And because of the that is actually not a wear and tear, it's actually a dysregulation. There are several targets you can actually identify for future treatments. And improved diagnosis will probably be there in the coming years. So we actually can have a surrogate model. So if we'll go back to the first picture, 270 elated, do we have a treatment? We are not there, but we are getting closer. And of course, there are numerous of persons I would like to thank that is from my group. And also, I would like to say, when we were in the beginning, we had good allies in the UK by George Bentley and we have James Richardson in Auschwitz. The people from Finland, Ilkka Kiveranta, they were quickly adopting this. In our own hospital, we have never treated one single patient because they didn't like the idea. But we treated a lot of other patients at a nearby hospital. So I thank you for this story. Thank you.