Accelerating Research on Intractable Diseases with "Organoids," Miniature Living Organs

2019/12/20

Throughout human history, the types of prevalent diseases have changed dynamically. In the modern era, not only cancer, which has become a national disease, but also inflammatory bowel diseases such as ulcerative colitis, once considered rare, are affecting an increasing number of people each year. The establishment of effective treatments is eagerly awaited.

A research team led by Professor Toshiro Sato of The Sakaguchi Laboratory (Organoid Medicine) at the Keio University School of Medicine has published research findings in the scientific journal *Nature* that delve into the mysteries of ulcerative colitis, a disease for which a curative treatment has not yet been found and whose mechanisms are not well understood. Thanks to the organoid culture technology first developed by Professor Sato and his team, the origins of various diseases that previously had no cure are now being unraveled.

The term organoid is a portmanteau of "organ" and "-oid," referring to a cluster of cells cultured to form a three-dimensional structure similar to that seen in living cells. In a sense, they are like small, living organs grown in a culture dish. Using these organoids, it has become possible to study the mechanisms by which normal cells change, unlike studying cells after a disease has already developed.

Ulcerative colitis is a disease in which the mucous membrane of the large intestine becomes inflamed for unknown reasons, leading to ulcers and sores (erosions). In many cases, symptoms include bloody diarrhea, bloody stools, and abdominal pain, which repeatedly worsen and then subside. This necessitates long-term treatment, and it is designated as an intractable disease by the Ministry of Health, Labour and Welfare. Although it was once a rare disease, the number of patients in Japan has recently increased to about 170,000 (total number of medical care beneficiary certificates and registrant certificates issued as of the end of fiscal year 2013).

Professor Sato's research team, using organoids to study ulcerative colitis, was the first in the world to reveal that colonic epithelial cells in inflamed areas accumulate specific genetic mutations that make them insensitive to the pro-inflammatory cytokine (a type of protein) interleukin-17 (IL-17).

The physicist Feynman said, "What I cannot create, I do not understand." I believe the same could be said for diseases. For example, in human research, we typically collect cells from a patient's lesion to observe and study them, but that is merely looking at the result of the disease. In the case of inflammatory bowel diseases like ulcerative colitis, for instance, various immune responses cause inflammation and destroy intestinal cells. However, just by looking at this ravaged intestine, we cannot understand how the originally normal intestine became such a wasteland.

After graduating from the Keio University School of Medicine and researching inflammatory bowel disease at the same university's graduate school, Professor Sato began studying intestinal regeneration. He studied abroad at the Stowers Institute for Medical Research in the US in 2005 and the Hubrecht Institute in the Netherlands in 2006. After returning to Japan, he also conducted research on gastrointestinal cancer.

"At the Hubrecht Institute, Dr. Hans Clevers was just in the process of publishing a paper on his discovery of intestinal stem cells. I was researching how to culture these intestinal stem cells and succeeded in identifying the three factors necessary for their culture. This stem cell culture technology that we developed was later found to be applicable to many other human organs and even across species."

Until now, genetic research has primarily relied on animal studies, mainly using mice. However, there is a growing global movement to reduce animal research and shift the focus to organoid research. Numerous organoid-related conferences are now held worldwide. This shift is also widely welcomed by society from an animal welfare perspective.

Currently, Professor Sato is culturing organoids of colonic epithelial cells to study the mechanisms by which normal cells develop into ulcerative colitis and colorectal cancer.

"We are literally 'creating' cancer and ulcerative colitis using organoids. Typically, when cancer or inflammation is present, we classify various diseases based on visual observation and then analyze their genes for research. However, by using organoids, we can now observe how normal cells change as living organisms. We are verifying the process by which normal colonic epithelial cells become diseased. We have found that normal colonic epithelial cells not only require a very high-quality and rich variety of 'cell food' (such as growth factors) to grow, but their growth area is also limited to a specific location on the basement membrane. On the other hand, epithelial cells from colorectal cancer have weed-like characteristics, capable of growing with very few types of 'food,' even in the deep parts of the intestine, muscles, or even other organs where intestinal epithelial cells normally cannot grow. It was considered possible that as the 'food' necessary for survival gradually decreased, the fragile normal cells transformed into cancer cells that can survive with nothing."

Furthermore, the existence of new factors that cause cells to become cancerous has also come to light.

"For example, when we try to create cancer cells from normal cells, it has become clear that cancer does not form based on conventional wisdom alone. When we introduce four or five genetic mutations known to be specific to cancer cells into normal colonic epithelial cells, they do indeed become weed-like cells that can grow with little food. However, that alone did not turn them into genuine cancer cells with the characteristics of invasion and metastasis. By trying to create them, we discovered for the first time what was similar but still missing. We are now going back to re-examine the additional factors needed, continuing the process of getting closer and closer to real cancer cells."

Research using organoids has also made the aging phenomenon of stem cells visible. In our bodies, cells are constantly being born, dying, and being replaced by new cells. It is stem cells that produce these new cells. Since clones are born from multiple stem cells, it was previously impossible to study only the clones born from a single stem cell. However, with the intestinal epithelial stem cell organoid culture technology developed by Professor Sato and his team, it is possible to proliferate numerous clones from a single stem cell. Using this method, Professor Sato has also reported research that used genome sequencing to investigate how many genetic mutations occur per single stem cell.

"Stem cells divide repeatedly, and each time, their genes are replicated. However, there are hundreds of millions of stem cells in the intestine, and with each daily division, replication errors—that is, genetic mutations—gradually accumulate. Our research has revealed that in the human body, approximately one genetic mutation occurs every week, and about 40 occur per year. This happens to everyone and is unavoidable. By counting the number of mutations, we can determine the so-called 'genomic age' of the cells. Since mutations accumulate over time in most of our body's cells, the number of mutations increases with age. For example, we know that a 40-year-old's colorectal stem cells have about 3,000 mutations. Therefore, if a colorectal stem cell has 3,000 mutations, its intestinal genomic age can be estimated to be 40, regardless of the person's actual age."

Next, as a first step in searching for factors that affect genomic age, Professor Sato examined the impact of inflammation in ulcerative colitis on genomic age. This led to the discovery of the previously unknown genetic mutations in ulcerative colitis.

"Disease does not have a clear boundary; it progresses very gradually. This research focuses on the invisible changes in its very early stages. In ulcerative colitis, there are cases where inflammation spreads to only one side, left or right. This time, we used organoids to compare the genetic mutations in normal epithelial cells and epithelial cells from the inflamed area of the same patient. The results showed that while there were indeed more genetic mutations in the inflamed area compared to the normal epithelial cells, the difference in number was not as large as expected. However, when we examined the types of mutations, it became clear that the inflamed cells in ulcerative colitis had characteristic genetic mutations that were previously unknown."

Cells communicate by secreting proteins called cytokines, which send various signals to other cells. In ulcerative colitis, inflammation spreads, and during this time, many pro-inflammatory cytokines, which work to promote inflammation, are secreted.

According to Professor Sato, this state of high pro-inflammatory cytokines is originally a very inhospitable environment for normal colonic epithelial cells.

"Normal colonic epithelial cells are not usually exposed to pro-inflammatory cytokines; on the contrary, they would die if exposed. However, we found that in the epithelial cells in the inflamed areas of ulcerative colitis, genetic mutations had accumulated that made them unresponsive only to interleukin-17 (IL-17), a type of pro-inflammatory cytokine that sends the message 'Promote inflammation!' While genetic mutations accumulate in our stem cells with age, these mutations are normally random and diverse. But when we looked at the intestines in ulcerative colitis, they were filled with cells that had mutations in specific genes. This suggested the possibility that in ulcerative colitis, normal cells that are vulnerable to the inflammatory environment decrease, while cells that have mutated to withstand inflammation increase—that the epithelial cells are gradually replaced by cells with genetic mutations that block IL-17."

However, this accumulation of mutations is not directly linked to cancer development, he says.

"For research physicians like us, the real thrill of medical research lies in elucidating why and how diseases occur. Even if the colonic epithelial cells themselves become resistant to inflammatory signals, if an environment where such signals are constantly flying around persists for many years without a response, the overall inflammation of the intestine may worsen. We hope to continue further research and eventually reveal the full picture."

Ulcerative colitis is known to be prone to progressing to colorectal cancer. Does the accumulation of genetic mutations discovered this time have a positive or negative effect on the overall inflammation of the intestine? And how does the change to cancer arise from this? The new discoveries made possible by Professor Sato's organoid technology are leading to one new question after another, sparking research around the world that delves into the origins of diseases. A new door is now beginning to open in the study of intractable diseases whose causes were previously completely unknown.

Toshiro Sato
Graduated from Keio University School of Medicine in 1997. Ph.D. from Keio University Graduate School of Medicine in 2004. Postdoctoral Fellow at the Stowers Institute for Medical Research (USA) from 2005. Postdoctoral Fellow at the Hubrecht Institute (Netherlands) from 2006. Project Associate Professor, Department of Gastroenterology and Hepatology, Keio University School of Medicine in 2013. Associate Professor, Department of Gastroenterology and Hepatology, Keio University School of Medicine in 2016. Professor, The Sakaguchi Laboratory (Organoid Medicine), Keio University School of Medicine from 2018.