A New Window into Disease:
Organoids Accelerate Intractable Disease Research

The world organoid (organ + -oid [From Greek -oeidēs "resembling," "form of"]) refers to a three-dimensional mass of cultured tissue produced in vitro. Think of it as a miniature version of an organ grown in a petri dish. Using these organoids, researchers can study the mechanisms that cause normal cells to mutate, which is entirely different from studying cells after they fall victim to disease.

Ulcerative colitis is a disease in which the mucous membranes of the colon cause an inexplicable inflammation, resulting in ulcers and sores. In many cases, patients experience repeated flare-ups of abdominal pain or blood in their stool or diarrhea. Ulcerative colitis requires long-term maintenance treatment and has been officially designated as a specified rare and intractable disease by the Ministry of Health, Labour and Welfare. Though it was once considered a rare disease, in recent years, the number of ulcerative colitis patients in Japan has ballooned to nearly 170,000, based on the total number of medical care certificates and registrant certificates issued at the end of FY2013.

Using organoid cultures to study ulcerative colitis, Prof. Sato’s research team has shown for the first time that inflamed colonic epithelial cells accumulate a specific gene mutation that is insensitive to interleukin 17 (IL-17), a cytokine (protein) that promotes inflammation.

"Physicist Richard Feynman famously said, ‘What I cannot create, I do not understand.’ I think the same can be said for disease. In human trials, for example, cells are usually harvested from lesions in patients, but that only allows us to observe a certain condition. In an inflammatory bowel disease like ulcerative colitis, various immune responses cause inflammation that can destroy the intestinal cells. However, we can’t understand how a once-healthy intestine was transformed from a vibrant landscape to barren wasteland by observing a ravaged intestine alone."

Upon graduating from the Keio University School of Medicine, Prof. Sato remained at Keio to study inflammatory bowel diseases as a graduate student. He then began his research into bowel regeneration, going abroad to the Stowers Institute for Medical Research in the United States in 2005 before working at the Hubrecht Institute in the Netherlands in 2006. After returning to Japan, he went on to study gastrointestinal cancer.

"I went to the Hubrecht Institute just as Dr. Hans Clevers was compiling a paper on his discovery of intestinal stem cells. I studied how intestinal stem cells grow and succeeded in identifying the three factors needed for a successful culture. The subsequent stem cell culture technology that was developed, over time, proved to have implications for organs beyond the intestine."

Until now, genetic research has relied heavily on animal studies using mice. But with a mounting movement to reduce animal studies and excitement surrounding future research potential, the topic of organoid technology is sweeping medical conferences around the world. For those concerned with animal rights, this movement is a most welcome one.

Prof. Sato is currently culturing organoids from colon epithelial cells and is researching the mechanism by which normal cells mutate into diseases like ulcerative colitis and colon cancer.

"We are using organoids to try and ‘create’ cancer and ulcerative colitis. With cancer or inflammation, we have typically had to rely on visual observation to classify and name things. But by using organoids, it is now possible to observe normal cells as living organisms to see how they change and mutate. We are currently examining how normal colon epithelial cells become sick. So far, we have found that normal colon epithelial cells do not grow without a wide variety of ‘cell foods,’ such as growth factors, and also that the growth area is limited to a fixed place on the basement membrane. In contrast, human colon cancer epithelial cells grow like weeds deep in the intestines, in muscles, and even in other organs where intestinal epithelial cells would normally not grow. And they do this with limited cell food, in terms of type and supply. It was thought that with a decreased amount of food to survive, weaker normal cells might mutate into cancer cells that can survive without any food at all."

The team’s findings have also uncovered the existence of new factors that cause cells to become cancerous.

"For example, when attempting to create cancer cells from normal cells, it became clear that cancer could not be created on conventional terms. If we introduce four or five gene mutations known to be specific to cancer cells into normal colon epithelial cells, they will indeed give rise to cells that grow like weeds even with little food. However, that alone did not create true cancer cells with properties like infiltration and metastasis. It was not until we attempted to create these cells that we understood what was missing. We are now once again going back to examine the necessary factors, and we are finally getting closer to creating actual cancer cells."

Research using organoids has also led to an understanding of aging in stem cells. The cells in our body are in a constant dance of life and death, old cells continually being replaced by new ones. Stem cells are what allow these new cells to grow. But because multiple stem cells are required to produce a clone, it was not possible to study clones generated from a single stem cell. However, the organoid culture technology of intestinal epithelial stem cells developed by Prof. Sato and his colleagues allows a large number of clones to grow from a single stem cell. This increase in quantity has also allowed Prof. Sato to use genomic sequencing to report on the amount of genetic mutation found within a single stem cell.

"Stem cells are capable of dividing themselves repeatedly, and DNA is replicated with each division. However, DNA replication errors and genetic mutations are not uncommon in the process as hundreds of millions of adult intestinal stem cells divide and replicate each day. Our research has revealed that errors and mutations can accumulate within the human body at a rate of about one per week—around 40 per year. We also know that these gradual, random mutations are unavoidable and happen to everyone. Counting the number of mutations, you can figure out the ‘genomic age,’ or genome instability, of a cell. Mutations accumulate across most of the cells in our bodies over time, so the number of mutations naturally increases with age. For example, stem cells in the colon are found to contain approximately 3,000 mutations by the age of 40. Therefore, if the number of mutations in the colon stem cells is 3,000, we could estimate the genomic age of that colon to be 40 years old."

To find the factors that affect genomic age, Professor Sato then looked at the effect that ulcerative colitis has on cells, which led to the discovery of previously unknown genetic mutations associated with the condition.

"There is no clear demarcation with a disease, which progresses gradually over time. This particular study focused on the almost invisible changes that take place in the very early stages of ulcerative colitis. In some cases, inflammation only spreads to one side of the colon. In this study, we used organoids to compare genetic mutations in a group of patients between their normal epithelial cells and epithelial cells from the inflammatory environment. While the inflammatory environment contained many more genetic mutations compared with normal epithelial cells, there were not as many as we had expected. However, when we examined the types of mutations that took place, we found that the inflammatory cells in ulcerative colitis had a characteristic type of genetic mutation that was previously unknown."

Cells secrete cytokine, small proteins that have a specific effect on communication between cells through something called "cell signaling." Inflammation spreads with ulcerative colitis through the secretion of inflammatory cytokines, which promote inflammation.

Prof. Sato explains that it is difficult for normal colon epithelial cells to live in an inflammatory environment with so many cytokines.

"Normal colon epithelial cells are not exposed to inflammatory cytokines and can die if they are. However, we found that there is an accumulation of genetic mutations that causes epithelial cells in the inflammatory environment to not respond to interleukin 17 (IL-17), one of the cytokines that causes inflammation. As we age, our stem cells become loaded with genetic mutations, which are normally random and highly varied. However, when we looked at the mutations detected in a colon with ulcerative colitis, we found that the colon was full of cells with one specific genetic mutation. From this, we were able to deduce that ulcerative colitis causes a reduction in the number of normal cells that are vulnerable to an inflammatory environment. The only cells that survive and proliferate are those that can withstand the inflammation. We thought that epithelial cells might gradually be replaced by these mutated cells that block IL-17."

However, Prof. Sato says that there is no direct link between the accumulation of mutations and the onset of cancer.

"The real thrill of medical research for physician-scientists like myself is getting to the bottom of how and why diseases happen. Even if the epithelial cells in the colon become more resistant to inflammatory signals, inflammation may worsen throughout the intestinal environment if inflammatory signaling continues for many years without cells responding to it. I hope that my future research will continue to help us see the full picture of what is happening."

Ulcerative colitis is known to be a precursor to colon cancer, but what roles do the genetic mutations discovered by Prof. Sato have in inflammation found throughout the intestine? Do these mutations have a positive impact or a negative one? And how does this lead to the development of cancer? The discoveries made possible by Prof. Sato’s organoid culture technology lead to more and more new questions, generating research into the origins of disease all around the world. This technology has only just begun to open a new window into the causes of intractable diseases that were until recently impossible to study.

Professor Toshiro Sato
Toshiro Sato completed his Doctor of Medicine (M.D.) at the Keio University School of Medicine in 1997. He then went on to complete a Ph.D. at the Keio University Graduate School of Medicine in 2004. He then served as a postdoctoral fellow at the Stowers Institute for Medical Research in the U.S. and the Hubrecht Institute in the Netherlands in 2005 and 2006, respectively. In 2013, he returned to the Keio University School of Medicine as a Project Associate Professor at the Department of Cardiology and became Associate Professor in 2016. Since 2018, he has served as a professor of organoid medicine at the Sakaguchi Laboratory, Keio University School of Medicine.

This article was originally published on the Keio University School of Medicine website.
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