Stem cell scientist sees obligation to face ethical issues

While they are only a few millimeters in size, they are predicted to achieve great things in the future of medicine: organoids. The artificially cultivated microstructures are now being used in laboratories around the world. Here in Austria, the Institute for Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), which Jürgen Knoblich has chaired as scientific director since 2018, is busily working on the next breakthrough in organoid research.

medinlive: Mr. Knoblich, let's take a glimpse inside the laboratory. How is an organoid made?

Knoblich: Basically, there are two methods for creating organoids. One method has been developed in the laboratory of molecular geneticist Hans Clevers at the Hubrecht Institute in the Netherlands. This method uses adult stem cells (stem cells found in our organs, ed.) to mimic the regeneration of these organs in cell culture. Through this regeneration pathway, the organs can be built. This method is relatively simple and applicable in large quantities, but it has two major disadvantages: First, it can only be applied to patients from whom a tissue sample has been previously taken. Second, the method does not work with organs that cannot regenerate. However, the development of the human brain and the development of diseases cannot be studied with this method.

That is why we at IMBA have developed a method that produces organoids from induced pluripotent stem cells (iPS cells, body cells, for example from a blood or skin sample, that can be reprogrammed into stem cells according to a method by Shinya Yamanaka, ed.). In this method, you switch on four genes in the lab, which causes the cells to go backwards in their development, they develop back to the state they had just before fertilization. Then you can make those cells advance again in the lab and turn them into any cell in the human body. This is possible in the cell culture dish; in our lab, we recreate the development of an organ three-dimensionally in this cell culture. For example, kidney, lung and heart organoids can be created.

medinlive: Do organoids differ from human brains?

Knoblich: An organoid will always differ from a human brain; that has to be said clearly. We start with pluripotent iPS cells and generate a neuroectoderm based on them (the neuroectoderm is the part of the outer cotyledon/ectoderm from which the nervous system develops during embryonic development, note). Then the organoids are put into a substance called Matrigel (a complex mixture of biomolecules used in 3D cell culture as a basis for growth - matrix, cell substrate - note), where they develop. There, the organoids reach a stage where histologically they look like a human brain, but anatomically they do not. The whole organoid does not look like a brain, but if I look at just a small region, it is remarkably similar to the human brain. This similarity persists until about the end of the first, beginning of the second trimester of fetal development. From that point on, organoids become increasingly dissimilar to a real brain.

Nevertheless, organoids can be cultivated for several years. During this time, the cells grow more and more mature and undergo the same maturation processes that a human nerve cell undergoes during embryonic development. In this regard, data now shows that it is possible to control this maturation process of the neurons until the end of embryonic development. But after a year, an organoid looks nothing like a human brain - it's a ball of cells, like a cabbage and turnip. Still, it's useful because I can study what's happening in a patient: Do the cells have the correct connections? Are they sending their axons in the right direction, are the regions wired up properly? In any case, an organoid is not a human brain, but a model of a human brain, a three-dimensional culture that has the characteristics of a human brain. The term "mini-brain" that is often used is very misleading. And the important thing is that we don't need a whole human brain for our research either. We can already study neurological diseases.

medinlive: Using the example of autism patients: What could organoid research look like in detail?

Knoblich: You take about 20 autism patients and 20 healthy patients, produce organoids from their iPS cells and then examine how they are composed cellularly, which cells occur where and which genes are switched on. This can now be done in bulk, 20,000 cells in one shot, which can be analyzed using a process called single cell sequencing. I expect to see very significant progress in this area in the coming year.

medinlive: What impact do you think organoid research has on medical treatment?

Knoblich: Organoid research is still relatively new, approximately ten years old, and has not reached the clinic yet. There is no such thing as an organoid therapy yet, even though there are already very good results in research. For now, the value of organoid research is primarily in research and in the fact that we can study disease mechanisms directly in humans, such as schizophrenia or autism, about which we know very little. Medical research over the past 30, 40 years has been very much focused on animal models. While we can cure cancer 100,000 times in a mouse, and that has taught us a lot, there has been a gap in our knowledge of all the processes that occur only in humans and not in animals. These include, for example, processes that take place in the brain. Our brain develops fundamentally differently from that of a mouse. We can now study these processes in organoids. I expect to make some big discoveries in the next five to ten years; organoids will have a real effect.

Another big advantage of organoids is that the genetic background is preserved in the iPS cells during reprogramming, which can never be replicated in an animal model. This allows me to determine, for example, whether a particular drug is effective or not in a specific patient.

medinlive: When will organoid research get to patients? What is the role of organoids in drug development?

Knoblich: In the last two to three years, the first companies have been founded that use organoids to develop drugs. It is difficult to say how long it will take to develop such a drug. You can assume that it takes about ten years to develop such a drug and then you have to allow another five to six years for the final clinical trials.

There are already examples of organoids being used in the clinic. One famous example comes from the Netherlands and the laboratory of Hans Clevers. There, research was conducted on patients with cystic fibrosis. The cause of the disease is a genetic defect called CFTR. While available drugs work for most patients, there are a few patients who have a different defect in the same gene and who would need different drugs. Drug development for this small number of patients would be prohibitively expensive and health insurance would not pay for it. Therefore, a program was started in which a biopsy was taken from all patients suffering from this rare genetic defect in the Netherlands, and from this sample intestinal organoids were made and drugs were tested. If the organoid therapy works, patients will be treated with it and the health insurance company will pay. Organoids are being used clinically, but there is no widely used therapy yet.

medinlive: Are there any studies comparing organoid models and animal models?

Knoblich: As far as research on the brain is concerned, the application is a 'no-brainer'. That was also mentioned in our original publication. But organoids can also be made from animals. In the meantime, there is an entire zoo. Organoids from orangutans and other primates or macaques can be used to learn how the evolution of our brain took place. After all, this is still a black box. We don't know what makes our brain human and what happened to make our brain so powerful. This is something that has not yet been studied. Now we can investigate which specific genes in humans are responsible for what. The research is also still in its early stages, but some breakthroughs will come in this field in the next years.

medinlive: What methods can be used to make brain organoids larger, more complex and more durable?

Knoblich: There are three different methods: First, we can mix organoids with other missing cells, such as blood vessel cells or microglial cells. The second method is bioprocess engineering. There are promising approaches to keep organoids in culture dishes containing microchannels, where I can add growth factors on one side and not on the other. The third method is fused organoids. For example, if I want to recreate neural pathways that run from the midbrain to the cerebrum, I can make them separately and fuse them. Organoids, after all, like to stick together. Then I'll see that the nerve fibers fuse together. The most famous example comes from Madeline Lancaster (a former IMBA researcher who now has her own lab in Cambridge/UK, note). She established this methodology in my lab. She made a cerebrum organoid and co-cultured it with spinal cord and muscle (prepared from rats). So she was able to show that the human brain tissue can make the rat muscle twitch. So the connections are absolutely in tact. We use this methodology, for example, to study spinal cord injuries.

medinlive: Who are the driving forces behind organoid research internationally?

Knoblich: The iPS cell research comes from Japan with the scientists Shinya Yamanaka and Yoshiki Sasai. Sasai started doing research on three-dimensional cultures. The first to start research on organoid diseases was Hans Clevers in the Netherlands. There is also a hospital in Ütrecht that specializes in this. We at IMBA were the first to show that three-dimensional cultures of iPS cells can be used to simulate diseases. At IMBA, five groups are now doing research on organoids. Our lab works with brain organoids, Josef Penninger's lab works with blood vessel organoids, one group makes heart organoids, another group makes so-called blastoids, which reflect the very first steps of human development, and one group makes intestinal and gastric organoids. Beyond that, globally, it's the usual suspects like MIT in Boston that have a focus on organoids. But the number of research groups working on organoids worldwide is in the hundreds. There are many countries that want to set up institutes in this field, China and the U.S. already have them.

medinlive: In an experiment, U.S. researchers connected organoids to a robot. They then used electrical impulses to stimulate the organoid to send signals to the robot, which then controlled it. What do you think of such experiments?

Knoblich: Such experiments make little sense in my opinion. The signals that are exchanged here could be random. What is useful, on the other hand, are methods for examining a large number of nerve cells simultaneously. To do this, we use microelectrodes, i.e. rods with up to a thousand electrodes attached, each of which can measure one or two nerve cells. You hook these up to a computer and then you can look at patterns of nerve cell excitation in an organoid. In any case, the most important thing in research is that after the research is done, you draw comparisons between healthy patients and sick patients and then draw conclusions about that disease.

medinlive: Moving on to the ethical debate: How can we know whether organoids can develop a consciousness if this topic itself has not been sufficiently researched in humans?

Knoblich: That is exactly the problem. We don't have a clear definition, but we know what it takes to develop consciousness. For example, there are experiments in which test subjects are shown images for milliseconds. Even if the images are not perceived consciously, they still have an influence on subsequent behavior. If I now fade in the images for longer, the test person reports back that he has seen the image, then he has become aware of it. Then the brain waves can be measured. This showed that if the subject was unaware of the image, the visual cortex was stimulated. As soon as the subject became conscious of the image, large-scale connections within the cerebrum were stimulated. What consciousness needs are connections over large regions of our brain, and I don't have those in an organoid. Everything that is assigned mechanistically to a consciousness is not present in an organoid.

To cite a mental experiment here: In epilepsy, we have surgery where patients have certain regions of the brain removed. These regions can be very large. Now one would have to consider: Is consciousness in the human being that is left, or has some of the consciousness now migrated into this probe that the surgeon has removed? No one will say that the consciousness has migrated into the sample, yet these pieces of tissue are many times, larger, more complex and better wired than an organoid. Those are the main arguments for me to say that the risk of an organoid developing consciousness is close to zero.

medinlive: As a consultant and member of research committees, you are part of the ethical debate on organoids. What questions are being discussed there?

Knoblich: I am currently working together with neurobiologists, philosophers, ethicists and lawyers at the German Academy of Sciences on a statement in which we ask ourselves the following question: What would have to happen in order for us to say, now is the time to stop research. The result may be: If someone makes an organoid that is a meter in diameter, or if an organoid starts to move. Of course, that's very unlikely. But I think even if we as scientists don't see any ethical concerns, if there are concerns in the population, we have to deal with it. That's our obligation and that's what we do, and I do it very actively both in Germany and here in Austria. In the committee of the International Society for Stem Cell Research (ISSCR), we are in the process of rewriting the guidelines for stem cell research so that they cover organoids very specifically and take into account the ethical particularities of these brain organoids. In addition to Europeans, the committee also includes Japanese, Chinese - which I think is particularly important, since China is the largest research area in the world - and US-Americans.

medinlive: How does the ethical debate in Europe differ from that in other countries?

Knoblich: In Europe, the debate is more profound, goes deeper and lasts longer. In the U.S., people think pragmatically about what rules there should be, whereas in Germany, the aim is to have a two-year process where the first question is, what is consciousness? Only after that has been defined there might be approaches to regulations. One is going directly from a phenomenon to a rule, and here in Europe, at least in the German-speaking world, one is starting to think about the foundation and the theoretical background. For me it is extremely interesting to live in both worlds.

medinlive: Finding a global ethical framework seems difficult then?

Knoblich: The committee is supposed to come up with very practical guidelines for practice that, after a poll, will be published one-to-one in scientific journals and become a requirement for being allowed to publish there. Ethical rules already exist. If you don't follow them, you can't publish, and if you can't publish, there's no point in doing research as a scientist. In this way, you can create rules in this area that have a certain validity worldwide. Of course, if someone comes up with the idea of creating CRISPR/Cas9 babies, as happened in China, that's a different issue. But it's at least good that the reaction from the international research community, especially from Chinese scientists, was so strong that the researcher is now in jail.

medinlive: Can you give some insights into what ethical rules are currently being discussed in this committee?

Knoblich: It's about patient consent, for example. It is not enough to tell the patient: We want cell material and are doing research on it. Instead, you have to say explicitly: We are making organoids. And then explain exactly what an organoid is. The parents of a child suffering from a severe neurological disease could say, for example, that the child's brain must not be replicated. Most patients are cooperative, see the benefit of our work, and therefore support this type of research.

Then the matter is about rules on how to deal with data, which must be anonymous. Right now, I could sequence the DNA in my lab and find out who the patient was, which is not allowed. There are also rules that say how human DNA data must be secured on the computer, much like a bank account. And it goes even further. One question we're not clear about yet: What do I do as a scientist when I find out that a particular drug might be effective for a patient? I can't go back there because everything is anonymous. For this, there should be a code that can be accessed by consensus of the researcher and the physician, whereby the physician can identify the patients again and carry back the results of the scientist. These are the ethical issues that really concern us.  

medinlive: Are rights for organoids or conservatorship part of the ethical debate?

Knoblich: No, but questions like: Who owns the organoid? This is a problematic issue, because for a long time the opinion was that a patient's cells always belong to the patient. In some countries, people are now moving away from that. For example, if I lose a hair on the street, which has my cells on it, I could sue the street sweeper who took this hair to the incinerator for personal injury. That is of course completely absurd! However, more and more iPS cells are being produced in research, so this is an important question. A fundamental principle of research is that others can replicate my experiments. But if I am not allowed to share the cells, it will result in the need for many patients and no one will be able to replicate experiments. In my opinion, it will be very important to build so-called biobanks, where cell lines from healthy and diseased patients are stored and available to all scientists worldwide. At IMBA, we are currently establishing such a biobank for Austria. With the help of the Medical University of Vienna, we are aiming for a special kind of patient consent for this, and it needs special ethics, because it will be virtually impossible if the patient subsequently says: I would like to have back all the cells that are circulating from me worldwide.

medinlive: There have been some breakthroughs at IMBA: 2013 with the development of the first brain organoids, 2018 with the world's first organoids for blood vessels. What are you currently working on?
Knoblich: We have succeeded in using organoids to show that it is also possible to reproduce interactions of brain parts that are located far away from each other. We have also created tumor organoids. At the moment, we have a great cooperation with AKH Vienna, where we are generating organoids for epilepsy. With this, we can fundamentally change the whole mechanism of a certain type of epilepsy. We have discovered new cell types that only exist in humans and not in animals. I can't give more details yet because the work has not yet been published, but hopefully the results will cause quite a stir.

Milestones in organoid research

In 2006, Shinya Yamanaka, a biologist at Kyoto University in Japan, opened a new path to the study of the human brain. He found a cocktail of four proteins that can turn ordinary skin cells into stem cells, which then have the potential to transform into neurons, muscles or blood cells. Based on this, other researchers learned to grow stem cells resembling miniature organs in a dish. Organoids make possible medical tests that would otherwise rely on animal experiments. In recent years, models of the brain, heart or kidneys, among others, have been made.

In 2013, researchers led by Jürgen Knoblich from the Institute of Molecular Biotechnology (IMBA ) in Vienna achieved another breakthrough. For the first time, they produced small, short-lived brain organoids (Lancaster and Knoblich, 2014). Brain organoids in which human cells form into brain-like structures in the Petri dish. They are used to study schizophrenia and autism, Parkinson's disease, and eye diseases. In 2018, the world's first blood vessel organoids were developed at IMBA, which now make it possible to study common diseases such as diabetes in a targeted manner. IMBA is the largest institute of the Austrian Academy of Sciences (ÖAW) and now enjoys a global reputation in stem cell research. The stem cell initiative at IMBA is funded by a grant from the Austrian Federal Ministry of Science as well as the City of Vienna.

Hans Clevers of the Hubrecht Institute on the use of organoids in cancer research (video)

Robert Vries of the Hubrecht Institute on the status quo in organoid technology (podcast)

Possible applications in medicine (PDF) Dr. Georg Weitzer, Center for Medical Biochemistry, Max F. Perutz Laboratories, Vienna BioCenter MedUni Vienna, 2018.

"Science Direct" article on the ethics of cerebral organoid research.

"New York Times" article on brain organoids

"The Guardian" article on the ethical debate of organoids

"New York Times" article on Alysson Muotri

Factsheet of the sciencemediacenter on research on mini-tumors