Hans Clevers (2016):
Replacement Organs from a Petri Dish
The focus of the research conducted by the Dutch biologist and physician Hans Clevers is on adult stem cells, which are capable of repairing damaged body tissue. In 2009 he developed a method for stem cells removed from the body to multiply in a practically unlimited manner. This makes it possible for organoids – such as tiny intestines, stomachs, and livers – to be bred in a Petri dish. These miniorgans are suitable not only for testing drugs, which will reduce the number of animal experiments that are needed, but also for replacing organs damaged by disease. In 2013 the Prize winner succeeded in genetically repairing stem cells in vitro that had been taken from the intestine of a patient with the hereditary illness mucoviscidosis and then culturing them to form healthy organoids. Clevers hopes to be able to use the funds from the Körber European Science Prize to employ gene therapy to free patients with hereditary liver defects from their suffering.
Replacement Organs from a Petri Dish
Text: Claus-Peter Sesín
Photos: Friedrun Reinhold
Whoever today undertook a global tour of the laboratories of stem cell scientists might imagine that they had gotten lost in a Frankenstein movie. They might see organs growing in dishes on the scientists’ tables. Some of the tiny clumps of cells, measuring just a few millimeters, are already viable early stages of intestines, stomachs, livers, kidneys, hearts, prostrate glands, and even brains. Do the scientists want to breed these miniorgans to full size in order to sew them together to make a monster like in Mary Shelley’s horror novel from 1818?
Such notions would mean the visitor is in the wrong movie. No less sensational, however, is what the scientists actually intend to do with the organoids. The miniorgans bred from the body’s own stem cells hold promise to trigger a medical revolution. Such organoids could soon replace defective intestines, livers, or stomachs. Instead of receiving a structure made of cold metal or of artificial material, or an exogenous organ from a donor, the patient receives a natural replacement organ that is practically identical with the diseased original in his body, just that it is healthy. The actual healing takes place outside the body. After their removal, the diseased stem cells undergo highly precise repair by means of the newest methods of genetic editing. Consequently, the genetically repaired stem cells grow in a Petri dish to a healthy organoid. After reimplantation, they can replace the damaged organ step by step. This novel version of gene therapy has already been successfully tested in mice.
One of the trailblazing pioneers of this new field of research is Hans Clevers, a Dutch biologist and physician. In 2009, he developed a standard procedure with which adult stem cells can reproduce in a practically unlimited manner ex vivo (i. e., outside the body). The resulting organoids function in vitro (i. e., in a Petri dish) similar to how the corresponding original organ does in vivo (i. e., in the body). The Prize winner’s discovery triggered a veritable boom. In the meantime, over 200 teams of researchers worldwide are culturing miniorgans from adult stem cells according to Clevers’s method.
The procedure functions both with healthy and with sick stem cells. This enables scientists to isolate the defective stem cells in the cancerous growths and to use them to cultivate living models of the tumors. This can even be done in several Petri dishes at the same time. Physicians are already in a position to test a different cancer drug in each of the dishes. After concluding the tests, they then give the tumor patient the medication that had worked best. “Instead of treating cancer patients with an unspecific form of chemotherapy, we can prescribe them the medication that their tumor organoids responded to particularly well in the laboratory”, according to Hans Clevers. In the meantime, even many pharmaceutical laboratories employ organoids. They use them to test new medications under very realistic conditions. If nothing else, this makes many animal experiments superfluous, and the results of that testing often cannot be transferred to humans one to one anyway.
“Instead of treating cancer patients with an unspecific form of chemotherapy, we can prescribe them the medication that their tumor organoids responded to particularly well in the laboratory.”
While he was an immunologist at the University of Utrecht, Hans Clevers worked primarily on the differentiation of white blood cells (T lymphocytes), which are created by hematopoietic, or bloodforming, stem cells in bone marrow. Later the focus of his research shifted to adult stem cells in the digestive organs, particularly in the small intestine. Adult means mature; adult stem cells as used here in contrast to embryonic stem cells – which are only present in early embryos – are present in the body at birth and can repair defects throughout one’s life. For example, if you cut yourself in the finger, then stem cells in the skin repair the defective tissue. In the small intestine, stem cells not only eliminate damage but also regularly renew its lining (epithelium). Stem cells in the small intestine are among the most active cells in the entire body.
As early as 1745, Johann Lieberkühn, a Berlin physician, used wax impressions to determine that fine invaginations extended out of the small intestine, which he called crypts. Viewed from the inside, the crypts are pits. These narrow pits are located between the fingershaped processes pointing toward the inside of the intestine. Among other things, they emit digestive secretions and absorb food. The intestinal stem cells sit at the base of the crypts. The daughter cells that arise by means of cell division migrate up the crypt walls, in the process differentiating into the six types of cells present in the intestinal epithelium. After leaving the crypts, the now mature intestinal cells continue step by step until they reach the peaks of the processes, where they die and make room for the following cells. This is the means by which the inner layer of cells of the human intestine is completely renewed approximately every six to eight days. Those of a mouse do this every five days. The strong propensity of adult stem cells to divide, however, also harbors a risk of cancer. A stem cell, if its genome has been damaged, such as by strong radiation or environmental toxins, can transform into a tumor cell.
When Clevers began his studies of the small intestine, he was surprised that previous research had been concerned almost exclusively with intestines that were sick, such as those stricken by cancer, and hardly with the regeneration of healthy intestines. He was interested particularly in the biochemical signals that stimulate the intestinal stem cells to divide as part of the routine renewal of the epithelium, but also in malignant growths. This is triggered by growth factors that dock on the receptors of the stem cells. After studying this for years, he found one receptor named Lgr5 that was only present on stem cells, and it was on this receptor that the growth factor R-spondin docks. Thanks to the Lgr5 marker, the intestinal stem cells can be easily isolated from sample intestinal tissue. Even stem cells of many other organs, such as the liver, stomach, pancreas, kidney, a woman’s breast, and prostrate glands in men, have Lgr5 receptors.
In 2009, Clevers achieved another sensation. Together with his Japanese postdoc, Toshiro Sato, he produced an intestinal organoid that survived for many months in a Petri dish, growing from a single intestinal stem cell. The ingredients that his team needed consisted in a cocktail made up of several growth factors and a gellike protein matrix, which served as a supportive frame making it possible for the organoid to expand in three dimensions. Scientists had previously performed such culturing experiments only in two dimensions.
The Prize winner has already proven that reimplanted liver organoids can actually perform the functions of a liver. This demonstrates in principle their suitability for serving as a replacement organ. One day this could make donor livers superfluous. Liver therapy in the future might consist of liver organoids – that were cultured from the patient’s own liver stem cells – being injected into the patient’s blood or directly into his liver. They would then gradually replace the diseased liver.
Liver organoids have the enormous advantage over donor livers that they are cultured from the body’s own stem cells. Consequently, there is no immune response rejecting an organoid that has been implanted. Clevers has already succeeded in confirming this experimentally in mouse experiments. Someone who, in contrast, receives a donor liver has to take immunosuppressive medication their entire life, and although this medication prevents a rejection, it also weakens the immune system and thus raises one’s susceptibility to diseases, such as infections. In the USA, every eighth death results from the failure of one’s own liver or of a donated one.
Hans Clevers wants to use the funds accompanying the Körber Prize to take the first steps in the direction of gene therapy. A procedure called CRISPR/Cas9 that was developed four years ago makes it possible to carry out precise repairs of defects in DNA, our genetic material. It even makes it possible to `edit´ individual nucleotides (letters) in DNA. Already in 2013 Clevers succeeded in correcting this gene defect in the intestinal stem cells of patients suffering from mucoviscidosis, a monogenic hereditary illness. Intestinal organoids cultured from the repaired stem cells showed in in vitro tests that they were healed of the mucoviscidosis. However, this was only a feasibility study, and no organoids were transplanted to patients.
This is a step that Hans Clevers will first dare to undertake in the future for patients suffering from two rare monogenic hereditary disorders of the liver, namely type 1 tyrosinemia and alpha-1 antitrypsin deficiency. In these patients, liver organoids in which the genetic defect has been corrected by using CRISPR/Cas9 are to be reimplanted following extensive safety tests, including a DNA analysis of the entire repaired genome. Clevers and his colleagues hope that the implanted repaired cells will gradually replace the genetically defect ones and that the patients will in this way be cured of their malaise. If a success, this would be the first gene therapy with cultured adult stem cells conducted on humans.
Organoids cannot only be cultured according to Clevers’s method, i. e., cultured from adult stem cells, but also from what are called pluripotent stem cells. The word `pluripotent´ means that these stem cells can differentiate to develop into any type of cell in the body. Pluripotent stem cells include embryonic stem cells. A novel technique even makes it possible to create pluripotent stem cells from normal adult somatic cells, such as skin cells. To do this, the adult cells are simply reprogrammed back to an embryonic stage. The result of this reprogramming is referred to as an induced pluripotent stem cell (iPS cells).
The iPS cells are, however, less well suited for culturing organoids for the purpose of transplantation because iPS organoids have until now exhibited a tendency to form tumors. “This is presumably a consequence of the intensive cell manipulations that have to be carried out to produce iPS cells”, comments Hans Clevers. “In contrast, it is very unusual for mutations to arise in organoids from adult stem cells, as DNA sequencing has shown”.
While adult stem cells may be less versatile that iPS ones, “They on the other hand are practically perfect at doing what nature created them for, namely the restoration of damaged tissue in their own environment”, Clever says. Another point is that adult stem cells that are already in a finished state can be isolated from excised tissue, while the creation of iPS cells can take up to several months. It has often taken this long for a sufficient number of iPS cells to form in many samples.
After their discovery, iPS cells were initially considered the epitome of stem cell research. Yet after Clevers had published his technique for the unlimited generation of adult stem cells in the science journal “Nature”, his procedure became the stronger focus of scientists and the scene for iPS cells became quieter. Many of Clevers’s colleagues in Utrecht chose an organ system that particularly interested them, to then continue their research on their own, frequently outside of the Netherlands.
For example, Sina Bartfeld, a biologist who was previously a member of Clevers’s team and today is at the Institute for Molecular Infection Biology in Wurzburg, is forming a working group for cultivating ministomachs. She wants to use them to study, for instance, the genesis of stomach ulcers. “It only takes two weeks until ministomachs are generated from adult stem cells”, Bartfeld raves. “The fascinating thing is that the stomach organoids organize themselves on their own and continue to grow. The culturing technique that Hans Clevers developed is an inexhaustible source of human cells of a certain tissue”, For her, it is an advantage that the adult stem cells do not need to be modified. “You can use them in the state in which they were removed”, according to Bartfeld. Furthermore, the culturing technique is simple to learn, and the miniorgans are easy to handle since it is no problem for them to be frozen, thawed, and sent by mail.
The possibility of freezing organoids is a feature that Clevers himself employs. In 2015, he began to create a comprehensive organoid biobank in which intestinal organoids from intestinal cancer patients are stored for research purposes in liquid nitrogen. Parallel to it, the genome data of each donor of stem cells are stored in a data base. The organoids and the respective genome information are available to research groups from around the world.
Hans Clevers and his colleagues collaborate with, for example, physicians at the Netherlands Cancer Institute in Amsterdam. Clevers’s team has cultured organoids from tumor tissue of intestinal cancer patients that had undergone chemotherapy there. The goal is to prepare prognoses of the actual healing process. “The oncologists treat their patients as they would otherwise”, Clevers says. “Parallel to that, we want to conduct laboratory experiments on the organoids to assess whether we could have predicted the outcome of therapy”. Clevers’s team also undertakes a study of patients suffering from advancedstage intestinal cancer. “For such patients there is no standard course of treatment”, he says.”We generate organoids from the tumor cells and test different drugs on them. We subsequently inform the physicians which medication worked the best”. The result is available relatively quickly, often within a few weeks.
In the context of his basic research, Clevers has above all been concerned with the mechanisms that lead stem cells to divide. An important role in this is played by the transmembrane receptors sitting on the cell membrane. They were given their name because a part of one sticks out like an antenna, while its root points toward the inside of the cell. Only very specific extracellular messengers can bind to the external antenna, and they fit like a key into the antenna’s lock. This key-lock principle prevents, as it were, false alarms. When such a messenger binds to the antenna, the root of the receptor releases secondary messengers inside the cell, triggering specific reactions that frequently proceed over several stages. Cell division is an example of such a reaction. Scientists refer to this chain of reactions as a signal cascade.
Stem cell division is a highly complex process. It requires several external messengers simultaneously docking at different receptors. One of these messengers is the growth factor R-spondin, which binds to the Lgr5 receptor that Clevers discovered. This bond strengthens the Wnt signal path, which Hans Clevers has studied in detail.
Wnt is also a growth factor. At the end of the Wnt signal path is a protein named beta-catenin. Although this factor is constantly being produced in stem cells, at the same time it is also being degraded, normally preventing Wnt from becoming active. This changes as soon as the growth factor Wnt docks to its receptor. Then the Wnt signal cascade starts inside the cell, inhibiting the degradation of beta-catenin. This leads to a strong increase in the concentration of this protein in the cell. The consequence is that beta-catenin now also penetrates into the nucleus and binds to the transcription factors, which trigger the process of reading out the genetic material DNA. The genes that are activated along the Wnt signal pathway determine, together with others, whether a stem cell continues to be a stem cell, whether it continues to divide, or whether it differentiates into another cell type.
A central component of the Wnt signal pathway is the tumor suppressor protein APC (adenomatous polyposis coli). This guardian protein prevents uncontrolled cell division and even plays an important role in tumor cells. Clevers and his colleagues have determined that the APC gene is mutated in almost all the malignant intestinal stem cells in patients with intestinal cancer. This defect prevents the APC protein from fulfilling its function as a guardian, the consequence of which being the uncontrolled cell division that is typical of cancer. “This is as if the intestinal stem cell were to constantly receive a Wnt signal to divide”, explains the Prize winner.
Hans Clevers has already been awarded many firstclass science prizes in honor of his pioneering discoveries. When he received the renowned Breakthrough Award, he invited the guests attending the ceremonies to a symposium at which scientists from his team recalled the research that the team had already conducted.
It was there that the Prize winner experienced something very revealing. “Based on my own recollection, the work at our laboratory appeared to be nothing but a success story. Looking back, everything seemed to have developed in a straight line. But as I listened to the research reports of my colleagues at the symposium it became clear to me: In reality the work in our laboratory was an endless succession of mistakes and errors. What we really did well was to spontaneously try out something that was promising, but also to give it up in timely fashion if we noticed that it was not leading to our goal and instead turn our attention to something else. In the rare moments we had the impression we had struck gold, we were willing to change our entire laboratory. We tried out many things, and it was only the ten percent that were successful that in hindsight had glorified my memories”.
“In molecular biology, we can conduct endless research; there are billions of effects to be discovered. There are often many different solutions to one problem. Evolution has however just picked one of them, and it would be arrogant to believe that we could reconstruct this in our minds.”
It is consistent with this that Clevers meanwhile considers science an art. The classical approach based on hypotheses is inappropriate. “In our team we proceed on the one hand very systematically, and our work is based on reproducible quantitative systems. But then we add something new to these systems, even purely experimentally, and observe impartially what happens. This is difficult psychologically since our brains tend to create causal relationships even then when there aren’t any. For this reason I constantly recommend that my team keep an open mind. We have to make observations without immediately making assumptions about what is probably taking place”.
Everything else is presumptuous, even with regard to evolution. “In molecular biology, we can conduct endless research; there are billions of effects to be discovered. There are often many different solutions to one problem. Evolution has however just picked one of them, and it would be arrogant to believe that we could reconstruct this in our minds”.
The prizewinner 2016
Hans Clevers, who was born in the Dutch city of Eindhoven in 1957, set up a provisional chemistry laboratory in the attic of his parents’ house while still a pupil: “I thought it was wonderful to mix the different substances together. I learned from my chemistry teacher how much fun it is to work in a lab”.
He studied biology and medicine and was awarded his doctorate in immunology at the University of Utrecht in 1985. In the following years, Hans Clevers wavered for a long time between striving for a career as physician or as a researcher. “The work of a physician is rewarded socially every day; you get a lot of positive feedback from patients. Research, in contrast, holds the promise of discovering uncharted territory, which holds just as much appeal to me”.
As a postdoc at the Dana-Farber Cancer Institute in Boston, he definitely made up his mind to pursue a career as a scientist. Upon returning to Holland, he accepted a position as professor at the University of Utrecht. From 1991 to 2002 he taught immunology, and after that molecular genetics. Parallel to this, he was President of the Netherlands Academy of Sciences from 2012 until 2015. Since 2015 Clevers has directed the research department of the Princess Máxima Center in Utrecht, a new hospital for treating children suffering from cancer.
Among Hans Clevers’s most important scientific achievements is the development of a procedure for the unlimited proliferation of adult stem cells. Nowadays, scientists around the world employ his method to create mini-organs (organoids). One day, these mini-organs could make organ donations superfluous and be suitable for use in novel forms of gene therapy.
Clevers, who has already received numerous awards, considers science to be a welcome challenge. “Research can be frustrating because you fail 90% of the time. Ideas prove to be wrong. Experiments fail. You have to have a personality that is satisfied to only experience really fantastic moments just a few times a year”.
In his free time, the Dutchman plays hockey, goes skiing, and has run the New York marathon on a number of occasions. And what would he have liked to become most of all if a career as a scientist had not worked out? “Then I would presumably have become a writer. That job is characterized by even more competition than that of a researcher, yet at the same time it is very creative. The perfect mixture for me!”
Award Ceremony 2016
Photos of the presentation of the Körber European Science Prize 2016 to Hans Clevers in the Hamburg City Hall. These photos are free to use in the context of news coverage with the credits Körber-Stiftung/David Aussenhofer given below.