The organoid architect – Science Magazine

Hans Clevers pioneered lab-built mini-organs that can serve as models ofdisease

A basic biologist at heart, Clevers says he never expected his findings tobenefit patients.

By her 50th birthday, Els van der Heijden felt sicker than ever. Born with the hereditarydisorder cystic fibrosis (CF), she had managed to work around her illness, finishingcollege and landing a challenging job in consulting. But Van der Heijden, who lives in asmall Dutch town, says she always felt a dark cloud hanging over myhead. When she began feeling exhausted and easily out of breath in 2015, shethought it was the beginning of the end.

Then she read a newspaper article about a child with CF named Fabian whose life had beensaved after scientists grew a mini-organ from a tissue sample snippedfrom his colon, one organ that CF affects. Doctors had used the mini-organ to testivacaftor (Kalydeco), a drug so expensive that Dutch insurers refuse to cover it withoutevidence that it will help an individual CF patient. No such data existed for Fabian,whose CF was caused by an extremely rare mutation. But his minigut responded toivacaftor, and he improved within hours of taking it. His insurance eventually agreed topay for the drug.

Van der Heijden's doctor arranged to have a minigut made for her as well; itresponded to a drug marketed as Orkambi that combines ivacaftor and another compound,lumacaftor. Within weeks after she began taking that combination, I had anenormous amount of energy, she says. For the first time ever, I feltlike my body was functioning like it should.

The life-altering test was developed in the lab of Hans Clevers, director of the HubrechtInstitute here. More than a decade ago, Clevers identified a type of mother cell in thegut that can give birth to all other intestinal cells. With the right nutrition, histeam coaxed such stem cells to grow into a 3D, pencil tip-sized version of the gut fromwhich it came. The minigut was functionally similar to the intestine and replete withall its major cell typesan organoid.

That was the start of a revolution. Clevers and others have since grown organoids frommany other organs, including the stomach, pancreas, brain, and liver. Easy tomanipulate, organoids are clarifying how tissues develop and repair injury. But perhapsmost exciting, many researchers say, is their ability to model diseases in new ways.Researchers are creating organoids from tumor cells to mimic cancers and introducingspecific mutations into organoids made from healthy tissue to study how cancer arises.And as Clevers's lab has shown, organoids can help predict how an individual willrespond to a drugmaking personalized medicine a reality. It is highlylikely that organoids will revolutionize therapy of many severe diseases, saysRudolf Jaenisch, a stem cell scientist at the Massachusetts Institute of Technology inCambridge.

For Clevers, the bonanza has come as a surprise. A basic biologist at heart, he says henever had real-world applications in mind. I was always driven bycuriosity, he says. For 25 years we published papers with no practicalrelevance for anyone on this planet.

ON A BRIGHT JULY MORNING at the Hubrecht Institute, Clevers listenspatiently to presentations during a weekly lab meeting. One postdoc presents data on herefforts to develop an organoid model for small-cell lung cancer; another reportsprogress on culturing hormone-secreting organoids from human gut tissue. Whenever theirresearch questions strike him as uninspired, Clevers urges them to be more ambitious:Why don't you pursue something you don't know? he asks.

Hans is capable of raising questions that are not contaminated by the anticipatedanswer, says Edward Nieuwenhuis, chairman of pediatrics at University MedicalCenter Utrecht (UMCU) and a good friend. He has a better nose than most forsniffing around and finding interesting stuff, says Ronald Plasterk, whoco-directed the Hubrecht lab with Clevers from 2002 to 2007 and is now the DutchMinister of the Interior and Kingdom Relations. That approach has earned Clevers manyawards. In June, for example, he was inducted into the Orden Pour le Mrite, anelite German order with just 80 members worldwide.

Clevers began his career studying immune cells as a postdoc at the Dana-Farber CancerInstitute in Boston. He landed his first job at UMCU's clinical immunologydepartment in 1989, where he quickly became department head. Most of the work wasclinical, such as leukemia diagnostics and blood work for transplants. But myresearch interests were always much more basic than the environment that I wasin, he says.

In early work, he identified a key molecule, T cell-specific transcription factor 1(TCF-1), that signals the immune cells known as T lymphocytes to proliferate. Later hefound that TCF-1 is part of the larger Wnt family of signaling molecules that'simportant not only for immune responses, but also for embryonic development and tissuerepair. In 1997, his lab team discovered that mice lacking the gene for one of thosesignals, TCF-4, failed to develop pockets in their intestinal lining called crypts. Soonafter, a study with Bert Vogelstein at Johns Hopkins University in Baltimore, Maryland,showed that TCF-4 also helps initiate human colon cancer. Fascinated, Clevers switchedhis focus from the immune system to the gut.

Inspired by a flurry of research on stem cells at the time, Clevers began hunting forintestinal stem cells. More than 50 years ago, researchers deduced that rodent cryptsproduce many cells that survive only a few days, suggesting some unidentified,longer-lived source for the cells.

After almost a decade of tedious experiments, Clevers's postdoc Nick Barker struckgold in 2007: He discovered that cells carrying a receptor named LGR5 give rise to allcells in mouse intestines and that molecules in the Wnt pathway signal those cells todivide. Barker later found LGR5-positive cells in other organs as well. In some, thecells were always active; in others, such as the liver, they multiplied only whentissues sensed injury.

At the time, culturing stem cells was notoriously hard, but after combing throughprevious lab experiments, another postdoc in Clevers's lab, Toshiro Sato, concocteda mix of growth factors that coaxed the gut stem cells to replicate in a dish. He hopedto see a flat layer of cells. But what emerged in 2009 from a single LGR5-positive cellwas a beautiful structure that surprised and intrigued me, says Sato,now at Keio University in Tokyo: a 3D replica of a gut epithelium. The structureself-organized into crypts and finger-shaped protrusions called villi, and it beganmaking its own biochemicals. A paper about the feat was rejected several times beforebeing published. Clevers recalls: No one wanted to believe it.

Soon, the lab began culturing LGR5-positive cells and growing organoids from the stomach,liver, and other organs. It was an exciting time, and I really felt like we wereon the frontiers of discovery, says another postdoc at the time, Meritxell Huch,now at the Gurdon Institute in Cambridge, U.K. But we certainly didn'tthink we were opening a new field.

CAPTIVATED BY STEM CELLS and their potential to regenerate tissues, otherlabs were starting to make organoids. A few months before Sato's 2009 paper,Akifumi Ootani, a postdoc in Calvin Kuo's group at Stanford University in PaloAlto, California, reported using a different strategy to grow gut organoids. Kuo'smethod starts with tissue fragments rather than individual stem cells and grows them ina gel partly exposed to air instead of submerged in nutrient medium. Around the sametime, Yoshiki Sasai of the RIKEN Center for Developmental Biology in Kobe, Japan,cultured the first brain organoids, starting not with adult stem cells but withembryonic stem cells. Other researchers grew organoids from induced pluripotent stemcells, which resemble embryonic stem cells but are grown from adult cells.

The various methods create different kinds of organoids, each with advantages anddrawbacks. Kuo's organoids contain a mix of cell types, which enablesobservation of higher-order behaviors such as muscle contraction, hesays. Because those organoids include stroma, a scaffold of connective tissue essentialfor tumor growth, they may prove better for studying therapies that target the stroma,such as cancer immunotherapy. Clevers's mix of growth factors grows organoidsconsisting primarily of epithelial cells, so his technique doesn't work for thebrain and other organs with few or no epithelial cells. Nor can his organoids be used totest drugs targeting blood vessels or immune cells because organoids have neither.

Both methods can generate organoids from individual patients, producing a personalizedminigut in just 1 to 3 weeks. (Although Clevers's organoids originate from adultstem cells, isolating those cells isn't necessary; culturing a tissue fragment withthe right nutrients is enough.) The methods are reproducible, and the organoids remaingenetically stable in culture; they can also be stored in freezers for years.

In 2013, Clevers and others founded a nonprofit, Hubrecht Organoid Technology (HUB), tomarket applications. Clevers first proposed using organoids for tissue transplants, saysHUB Managing Director Rob Vries. Studies showed that healthy organoids implanted in micewith diseased colons could repair injury. But we bagged the idea because therewere too many regulatory hurdles and the chance of success was low, Vriessays.

Cystic fibrosis patient Els van der Heijden received a new drug combination basedon organoid tests. Within weeks, I had an enormous amount ofenergy, she says.

The idea of enlisting organoids to treat CF came from Jeffrey Beekman, a researcher atUMCU who studies that disease. All Dutch newborns are screened for CF, and colon biopsysamples are taken from babies who test positive. The tissue is tested to gauge howdysfunctional the defective gene is and then stored. Growing organoids from thosesamples would be relatively simple, argued Beekman, who has since spearheaded theproject.

CF can arise from more than 2000 mutations in one gene, which cripple the ion channelsthat move salt and water through cell membranes. The disease affects all tissues, butthe primary symptom is excess mucus in the lungs and gut, causing chest infections,coughing, difficulty breathing, and digestive problems.

Ivacaftor and the combination drug lumacaftor and ivacaftor, both marketed by VertexPharmaceuticals in Boston, restore the ion channels' function. But the drugsdon't work equally well for everyone, and they have been tested and approved onlyfor people with the most common mutations, together accounting for roughly half of allCF patients. Vertex, which declined to answer questions for this story, has beenreluctant to spend millions on trials in patients with rare mutations because thepotential payoff is small. And with the price tagboth drugs cost between100,000 and 200,000 per year in Europehealth services andinsurance companies have been unwilling to pay for the medicines for people with thoseuntested mutations.

Van der Heijden falls into that category because only two other people in the Netherlandsshare her mutation. But when organoids grown from her gut were exposed to lumacaftor andivacaftor, the organoids swelled like normal gut tissue, a sign that the defectiveprotein was working and that salt and water were flowing through. The result helpedpersuade Vertex to give her the drug through a compassionate-use program, withoutpayment. (Regulatory agencies require her to be monitored in a clinical trial.) Her sideeffects included fatigue, nausea, and diarrhea, but after a few months, it wasas if someone opened the curtain and said, Look, the sun is there, come out andplay, she says. And I did.

In collaboration with Vertex, HUB has tested ivacaftor on organoids grown from CFpatients who had taken part in a clinical trial of that drug. The study confirmed thatorganoids can predict who will respond to the drug.

HUB has also tested ivacaftor on organoids from 50 patients with nine rare mutations. Onthe basis of the results, insurers agreed to pay for the drug in six more Dutchpatients, and Vertex is following up with the first clinical trial of ivacaftor in CFpatients with rare mutations. Meanwhile, HUB is building a biobank, financed by Dutchhealth insurers, containing organoids from all 1500 Dutch CF patients for testing bothexisting drugs and new candidates.

This is the next big thing in CF research, says Eitan Kerem, head ofpediatrics at Hadassah Medical Center in Jerusalem, who is building a similar biobankand has launched a trial in patients with rare mutations. Organoids are especiallyuseful because no great animal models for CF exist, Kerem says; ferrets and pigs aresometimes used, but they are expensive and not available to mostresearchers.

Drug and biotech companies are now striking deals with HUB to explore organoids in otherdiseases. The success with CF suggests that they can model other single-gene disorders,such as -1 antitrypsin deficiency, which causes symptoms primarily in the lungsand liver. Some companies are also testing failed drugs on organoids and comparing theresults with animal and clinical data, hoping to find ways to predict and avoid suchfailures.

CANCER IS ALSO a major target. By growing organoids from tumor samples,researchers can create minitumors and use them to study how cancer develops or to testdrugs. Soon after the minigut paper came out in 2009, David Tuveson, who heads thecancer center at Cold Spring Harbor Laboratory in New York, began prodding Clevers todevelop organoids for pancreatic cancer, which is notoriously hard to treat. Existingcell culture models were not very realistic, Tuveson says, and creating geneticallyengineered mice took up to a year, compared with up to 3 weeks for pancreatic cancerorganoids.

The organoids have already helped clarify new pathways that lead to pancreatic cancer,Tuveson says, and unpublished data suggest that they will help researchers predict whichtreatments will be most effective. He and Clevers are trying to make the organoidsresemble real cancer more closely by adding stroma and immune cells. The Hubrecht lab isalso involved in two trials to assess whether colon cancer organoids grown fromindividual patients can predict drug response.

Charles Sawyers of Memorial Sloan Kettering Cancer Center in New York City is trying tomake prostate cancer organoids, but he says they are finicky. Organoids from primarytumors generally don't grow; those from metastatic tissue sometimes do, but normalcells often outgrow cancer cells. They seem to need a lot of tender love andcare, and there is no method to the madness, says Sawyers, who has succeededwith only 20 patients so far.

Organoids can be used to study how pathogens interact with human tissues. In thislung organoid grown in Hans Clevers's lab, cells colored green are infectedwith respiratory syncytial virus.

But Sawyers discovered that he could easily grow organoids from normal prostatetissueit just works beautifully, he saysand then usegene-editing techniques such as CRISPR to study any cancer mutation he wants. Isthis a tumor suppressor gene? Is this an oncogene? Does it collaborate with geneXY? You can play the kind of games on the scale that you alwayswanted to, he says. As Kuo puts it, We can build cancer from the groundup.

Other cancer researchers want in, too. Tuveson received so many requests for organoidtraining that he began hosting regular workshops at his laboratory. In 2016, the U.S.National Cancer Institute launched a scheme to develop more than 1000 cell culturemodels, including organoids, for researchers around the world to use, together withCancer Research UK in London, the Wellcome Trust Sanger Institute in Hinxton, U.K., andHUB.

Using personalized organoids to treat cancer still faces hurdles. Organoid culture time,which varies by cancer, must be shortened, and the cost, a few thousand dollars perpatient, needs to come down. Also, cancers accumulate genetic mutations as theyprogress, which could mean that an organoid grown from a patient's cancer early onmight not reflect its later state. Nevertheless, from my perspective it'sthe most transformative advance in cancer research that I know of, Tuvesonsays.

If all of that excites Clevers, he rarely shows it. He avoids emotional language whilediscussing his research, preferring instead to describe and explain. Even close friendssometimes find his pragmatism puzzling. He talks about his research like someonetalking about screwing in a screw, Nieuwenhuis says.

Clevers says he gets his high from the satisfaction of finding somethingnovel, regardless of practical applications. Recent experiments, for instance,suggest that when an organ lacks LGR-5-positive cells, differentiated cells may be ableto dedifferentiate and repair tissuesa radical change from theone-way street toward specific identities that stem cells were thought to travel.Some organs may not have a professional stem cell at all, Clevers says,with a hint of wonder. But when asked how he felt when he saw his findings have profoundbenefits for patients such as Fabian and Els van der Heijden, he simply says, Idid not expect that.

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The organoid architect - Science Magazine