Press Release

February 25, 2021
Keio University School of Medicine

Development of Transplant Therapy Gives the Large Intestine Digestive andAbsorptive Functions Unique to the Small Intestine
Understanding and developing treatments for short bowel syndrome and other small bowel diseases
A joint research team at Keio University has developed a technology for creating a small intestinalized colon(SIC)—a large intestine with absorption unique to the small intestine and peristaltic functions. This was achievedby replacing the native colonic epithelium with ileum-derived organoids. (Note 1) The team was led by ProfessorToshiro Sato and Assistant Professor Shinya Sugimoto at the Department of Organoid Medicine, Sakaguchi
Laboratory, Keio University School of Medicine, and Project Professor Eiji Kobayashi at the Department ofOrgan Fabrication, Keio University School of Medicine.
The small intestine has unique hair-like projections called villi (Fig. 1), which play an important role in thedigestion and absorption of food. In contrast, the large intestine does not have these kinds of protruding structuresand can hardly digest or absorb nutrients. It has long been a mystery why these projection structures are createdonly in the small intestine. In this study, the team found that the epithelium inside the small intestine senses
streams of intestinal juice and creates villous structures, and they were successful in culturing a villous small
intestinal epithelial organoid. This discovery led to the development of a technology that enables the team togenerate a “small intestinalized” colon (SIC) by replacing colon epithelium with small intestinal epithelium usingorganoid transplantation. By transplanting this SIC into a rat short bowel syndrome model, the team has shown,for the first time, that the SIC has a therapeutic effect on rats with short bowel syndrome.
Patients who have had a large portion of their small intestine removed due to Crohn's disease, intestinal torsion,or severe enteritis in the neonatal period develop short bowel syndrome, and the inability to adequately digest
and absorb proteins, sugars, and fats leads to short bowel syndrome with poor outcomes. Intestinal transplantation
currently remains the only treatment option for severe short bowel syndrome, but due to the shortage of donors
and comparatively strong rejection response, the number of transplants remains low when compared to otherorgans. And so there has been a need for the development of regenerative medicine as an alternative treatment
for small intestinal transplantation, but until now it has been impossible to create complex organs including bloodvessels and lymph vessels that carry absorbed nutrients throughout the body. The SIC technology developed inthis study transforms a different organ into a needed one and takes us one step closer to rejection-free organtransplantation through regenerative medicine. This study has also elucidated part of the origin of the projection
structure of small intestinal villi, which is expected to lead to a better understanding of the pathogenesis of various
small bowel diseases.
The results of this research were published in digital edition of the British journal Nature on February 24 (GMT),
2021.

1. Research Background
A Long-Awaited New Approach to Small Intestinal Transplantation
The mucosa of the small intestine has the essential function of absorbing nutrients and water in order to sustain life.
The surface of the small intestine is covered with epithelial cells, and for efficient digestion and absorption, there
are numerous projections about 0.5 mm long called villi. Between the villi are indentations known as crypts. (SeeFig. 1) The base of the crypt contains intestinal epithelial stem cells, which are responsible for the regeneration
mechanism of the small intestinal epithelium and enable the fastest epithelial cell turnover in the body. Intestinal

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epithelial stem cells continually replicate themselves throughout life (self-renewal), differentiate into all types ofintestinal tissue cells (multipotency), and migrate from the crypt to the villi.

When a large portion of the small intestine is removed for diseases such as Crohn's disease, patients may develop
short bowel syndrome, a serious and chronic malabsorption disorder. In severe cases, patients experience oral
feeding difficulties, with intravenous nutrition given through a central venous catheter, which involves injecting
nutrients into the veins through a tube. As a result, serious complications such as catheter-associated infection andliver dysfunction can lead to significantly reduced quality of life or even loss of life. Treatments for such cases
include surgery to extend the length of the small intestine, and outside of Japan, treatment with GLP-2, a drug that
stimulates the growth of intestinal villi. (Not yet approved for use in Japan) However, all of these treatments areinadequate and currently the only viable treatment is a bowel transplant, in which a healthy small bowel istransplanted from a donor. However, because the small intestine is more susceptible to rejection than other organs,the graft survival rate is low, and in Japan, only 32 small intestinal transplants (13 living and 19 brain-dead), in alimited number of facilities, were performed on 28 patients in the 20-plus years between the first operation in 1996
and December 2019. And even if a transplant is successful, there are still concerns about rejection, infections
associated with the use of immunosuppressive drugs, and the development of malignant tumors, among other issues.
For this reason, the number of small bowel transplants has remained limited, contrary to the increase in the numberof transplants for other organs.
Currently, there is a great need for innovative new treatments for intractable small intestinal diseases for which there
is no definitive cure, and expectations for regenerative medicine are high. However, since the small intestine is anextremely complex structure that includes blood vessels, nerves, muscle layers, and lymphatic vessels involved in
the absorption of lipids—in addition to the structure of the crypt and villi—it is difficult to construct all of thosepieces in vitro, and until now the idea of using regenerative medicine to treat small intestinal diseases has not beenrealistic.
Replacing the colonic epithelium with intestinal epithelium tissue for a functional small intestinalized colon
A research team led by Professor Toshiro Sato and Assistant Professor Shinya Sugimoto has previously establisheda technology that allows for the long-term culturing of intestinal epithelial stem cells prepared from intestinal
epithelium as a three-dimensional mass of cultured cells known as an organoid (Sato T, et al. Nature 2009, Fujii M,et al. Cell Stem Cell 2018). Organoids are expected to have potential not only as a research tool for human epithelial
cells in vitro, but also as potential transplantation cells, since they are epithelial stem cells cultured directly fromhuman intestinal cells. However, as mentioned above, it has not been possible to create the human small intestine
itself from a collection of human small intestinal epithelial cells, and it was technically difficult to transplant normal
human intestinal epithelial cells. Recently, a research team has succeeded in developing a technique to construct
healthy human colon epithelium in mice by completely removing the colonic epithelium of immunodeficient miceand replacing it with human colonic epithelium (Sugimoto S, et al. Cell Stem Cell 2018). We wondered if we could
use this technology for regenerative purposes to replace the colonic epithelium with cultured small intestinal
epithelium in patients with short bowel syndrome. (See Fig. 2)

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2. Research Significance and Future Development
Xenotransplanted human small intestinal epithelium re-builds villus structures in mouse colon
In this study, the research team first removed the colon epithelium of mice and attempted to transplant human small
intestinal epithelium. As a result, the transplanted small intestinal epithelium formed a different villi structure fromthat of the transplanted colonic epithelium and showed expression of proteins related to digestion and absorptionthat are only found in the small intestine. The formation of microvilli, which is important for absorption, was also
observed in transmission electron microscope imaging. Furthermore, we confirmed that small intestine-specificlymphatic vessels, known as lacteal, which are important for fat absorption, are also formed in the large intestine
through the transplantation of small intestinal epithelium. This showed that the small intestinalized colon, withreplaced epithelium, can digest and absorb nutrients and transport them into the body through the lacteal.
(See Fig. 3)

Luminal flow instructs villus formation
On one hand, the villous structure of the human small intestinal epithelium transplanted onto the colorectal surface
near the anus of mice was immature and inadequate compared to the original. Luminal contents of the humanintestine passes through the small intestine in a liquid state, and as it moves through the large intestine, it absorbs
water and salts and solidifies into feces. (See Fig. 4) For this reason, we thought that the formation of villous
structures may not be promoted near the rectum at the end of the large intestine due to insufficient “flow” ofintestinal juice.

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The research team then cultured the organoids of the large intestine and the small intestine as a monolayer,
artificially agitated the culture overlay medium to create a “flow,” and further cultured the organoids in thisenvironment. As a result, the small intestinal organoids facilitated the formation of villus-like structures that
protruded toward the lumen. On the other hand, colon organoids showed no such changes and remained flat,indicating the existence of a unique mechanism of villus formation in the small intestinal epithelium that isdependent on this digestive “flow.” (See Fig. 5)

Demonstrating the use of small intestinal epithelial organoids to treat short bowel syndrome in rats
Based on these findings, the research team then conducted an experiment in rats to demonstrate that short bowel
syndrome can be treated using small intestinal organoids. In collaboration with Project Professor Eiji Kobayashi ofKeio University School of Medicine and Professor Yoji Hakamata of Nippon Veterinary and Life ScienceUniversity, we sought to create a small intestinalized colon transplanted using small intestinal organoids establishedfrom transgenic rat small intestinal cells that express a luminescent substance called luciferase and then conducttransplantation experiments in healthy rats.
First, a part of the large intestine was cut away while preserving blood flow in the nutrient vessels. (See Fig. 6 left)
During the surgery, the epithelium of the detached colon was removed and a small intestinal organoid wastransplanted to the site. The grafts were then sewn to the abdominal wall and kept in the rat's body while maintaining
blood flow until the epithelium began to grow and attach. (See Fig. 6 center) This technique allows the transplanted
small intestinal epithelial cells to remain free of fecal passage, allowing them to wait for the transplanted small
intestinal epithelial cells to become engrafted in the colonic epithelium without being shed, making it possible totransplant small intestinal organoids over a wide area.
However, when fixed to the abdominal wall, there was no “flow” on the surface of the small intestinal epithelium
following transplantation, so sufficiently matured villi did not form. Therefore, in rats in which the whole small
intestine has been resected to reproduce short bowel syndrome, the transplantation was performed in the terminal
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ileum, which is adjacent to the small intestine and is where “flow” exists in the intestinal tract. The structure andfunction of the graft formed by ileum-derived organoid that differentiated and matured in a “flowing” environment
following transplantation, and the effect of transplantation on short bowel syndrome, were compared between cases
using small intestinal epithelial organoids and cases using colonic organoids. (See Fig. 6 right)

As a result, survival time was prolonged in ileum organoid-transplanted rats, and we observed extensive engraftmentof transplanted cells and formation of villi in survivors. In contrast, we did not observe any prolongation of survivalin the colon organoid transplant group. Similar to the experiments in mice, the grafts in the small intestinal organoidtransplantation group showed formation of lacteal-like structures and blood vessels, as well as intestinal peristalsis
with neurotransmission, and we confirmed them to be functional small intestinal grafts with the ability to absorb
lipids, glucose, and peptides.
These results indicate that the small intestinal epithelium has the ability to change and redesign the structure of thetransplanted tissue. This strategy of transplanting a small intestinalized colon, in which small intestinal organoids
replace the colonic epithelium, is expected to be applicable in humans, who are larger than mice or rats, and isexpected to pave the way for the development of new therapies using organoids that do not require
immunosuppressive drugs. This research method can also provide new insights into the formation of villous
structures of the small intestinal epithelium, an important structure for digestion and absorption, and is expected toprovide clues to understanding the pathogenesis of various small intestinal diseases.

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3. Notes
This research is an Advanced Research & Development Program for Medical Innovation (AMED-CREST) of theJapan Agency for Medical Research and Development (AMED): “Understanding of Pathophysiological Processes
and Discovery of Medical Technology Seeds through Spatiotemporal Research of Tissue Adaptation and Repair
Mechanisms,” Research and Development Objectives: ”Dissecting intestinal fibrogenic diseases by a newly
developed 4D disease model system” (Principle investigator: Professor Toshiro Sato, Keio University School ofMedicine) as well as the following programs:- Japan Agency for Medical Research and Development (AMED)’s Research Center Network for Realization ofRegenerative Medicine: “Center for development of mucosal regenerative therapies for inflammatory bowel
diseases using cultured intestinal epithelial stem cells”- JSPS Grants-in-Aid for Scientific Research (KAKENHI) JP20H03746 and JP17H06176- The Mochida Memorial Foundation for Medical and Pharmaceutical Research- Keio University Academic Development Funds
4. Research Paper
Title: An organoid-based organ-repurposing approach to treat short bowel syndrome
Authors: Shinya Sugimoto, Eiji Kobayashi, Masayuki Fujii, Yuki Ohta, Kazuya Arai, Mami Matano, KeikoIshikawa, Kentaro Miyamoto, Kohta Toshimitsu, Sirirat Takahashi, Kosaku Nanki, Yoji Hakamata, Takanori Kanai,Toshiro Sato
Publication: Nature (online)
DOI：https://dx.doi.org/10.1038/s41586-021-03247-2
Glossary
Note 1: Organoid
Cultivated in the form of sheets using conventional cell culture technology. The term organoid refers tocultured cells grown as a three-dimensional structure using hydrogels as scaffolding and nutrients known as
growth factors. From a single stem cell, it is possible to create a tissue-like structure in vivo and infinitely
increase the number of healthy cells found in various tissues such as the stomach, small intestine, large
intestine, pancreas and liver. In a previous study, the research team succeeded in culturing human intestinal
epithelial organoids using a novel culture method that reproduced most of the differentiated cells found inliving tissues. This method was also used in this study. It is also possible to culture organoids in sheet form,as shown in Fig. 5.

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Inquiries regarding clinical research
Department of Organoid Medicine, Sakaguchi Laboratory, Keio University School of Medicine
Professor Toshiro Sato
Assistant Professor Shinya Sugimoto
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Project Professor Eiji Kobayashi
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