論文 - 佐藤　俊朗

Gut pathobionts underlie intestinal barrier dysfunction and liver T helper 17 cell immune response in primary sclerosing cholangitis

Nakamoto N., Sasaki N., Aoki R., Miyamoto K., Suda W., Teratani T., Suzuki T., Koda Y., Chu P., Taniki N., Yamaguchi A., Kanamori M., Kamada N., Hattori M., Ashida H., Sakamoto M., Atarashi K., Narushima S., Yoshimura A., Honda K., Sato T., Kanai T.

Nature Microbiology （Nature Microbiology）  4 （ 3 ） 492 - 503 2019年03月

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© 2019, The Author(s), under exclusive licence to Springer Nature Limited. Primary sclerosing cholangitis (PSC) is a chronic inflammatory liver disease and its frequent complication with ulcerative colitis highlights the pathogenic role of epithelial barrier dysfunction. Intestinal barrier dysfunction has been implicated in the pathogenesis of PSC, yet its underlying mechanism remains unknown. Here, we identify Klebsiella pneumonia in the microbiota of patients with PSC and demonstrate that K. pneumoniae disrupts the epithelial barrier to initiate bacterial translocation and liver inflammatory responses. Gnotobiotic mice inoculated with PSC-derived microbiota exhibited T helper 17 (T H 17) cell responses in the liver and increased susceptibility to hepatobiliary injuries. Bacterial culture of mesenteric lymph nodes in these mice isolated K. pneumoniae, Proteus mirabilis and Enterococcus gallinarum, which were prevalently detected in patients with PSC. A bacterial-organoid co-culture system visualized the epithelial-damaging effect of PSC-derived K. pneumoniae that was associated with bacterial translocation and susceptibility to T H 17-mediated hepatobiliary injuries. We also show that antibiotic treatment ameliorated the T H 17 immune response induced by PSC-derived microbiota. These results highlight the role of pathobionts in intestinal barrier dysfunction and liver inflammation, providing insights into therapeutic strategies for PSC.

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Modeling Human Digestive Diseases With CRISPR-Cas9–Modified Organoids

Fujii M., Clevers H., Sato T.

Gastroenterology （Gastroenterology）  156 （ 3 ） 562 - 576 2019年02月

ISSN  00165085

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© 2019 AGA Institute Insights into the stem cell niche have allowed researchers to cultivate adult tissue stem cells as organoids that display structural and phenotypic features of healthy and diseased epithelial tissues. Organoids derived from patients’ tissues are used as models of disease and to test drugs. CRISPR-Cas9 technology can be used to genetically engineer organoids for studies of monogenic diseases and cancer. We review the derivation of organoids from human gastrointestinal tissues and how CRISPR-Cas9 technology can be used to study these organoids. We discuss burgeoning technologies that are broadening our understanding of diseases of the digestive system.

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Human Intestinal Organoids Maintain Self-Renewal Capacity and Cellular Diversity in Niche-Inspired Culture Condition

Fujii M., Matano M., Toshimitsu K., Takano A., Mikami Y., Nishikori S., Sugimoto S., Sato T.

Cell Stem Cell （Cell Stem Cell）  23 （ 6 ） 787 - 793.e6 2018年12月

ISSN  19345909

　概要を見る

© 2018 Elsevier Inc. Cellular diversity that shapes tissue architecture and function is governed by multiple niche signals. Nonetheless, maintaining cellular diversity in human intestinal organoids has been challenging. Based on niche ligands present in the natural stem cell milieu, we establish a refined organoid culture condition for intestinal epithelia that allows human intestinal organoids to concurrently undergo multi-differentiation and self-renewal. High-throughput screening reveals that the combination of insulin-like growth factor 1 (IGF-1) and fibroblast growth factor 2 (FGF-2) enhances the clonogenic capacity and CRISPR-genome engineering efficiency of human intestinal stem cells. The combination equally enables long-term culture of a range of intestinal organoids, including rat small intestinal organoids. Droplet-based single-cell RNA sequencing further illustrates the conservation of the native cellular diversity in human small intestinal organoids cultured with the refined condition. The modified culture protocol outperforms the conventional method and offers a viable strategy for modeling human intestinal tissues and diseases in an in vivo relevant context. Sato and colleagues develop a modified culture condition for human intestinal organoids that improves the culture efficiency and maintains their long-term multi-differentiation capacity. scRNA-seq of human small intestinal crypts and organoids demonstrates that in vivo cellular diversity can be preserved in organoids cultured with the refined condition.

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Induction of differentiation of intrahepatic cholangiocarcinoma cells to functional hepatocytes using an organoid culture system

Saito Y., Nakaoka T., Muramatsu T., Ojima H., Sukeda A., Sugiyama Y., Uchida R., Furukawa R., Kitahara A., Sato T., Kanai Y., Saito H.

Scientific Reports （Scientific Reports）  8 （ 1 ）  2018年12月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  2045-2322

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© 2018 The Author(s). Intrahepatic cholangiocarcinoma (IHCC) is a highly aggressive malignancy with a poor prognosis. It is thought to originate from cholangiocytes, which are the component cells of intrahepatic bile ducts. However, as patients with viral hepatitis often develop IHCC, it has been suggested that transformed hepatocytes may play a role in IHCC development. To investigate whether IHCC cells can be converted to functional hepatocytes, we established organoids derived from human IHCC and cultured them under conditions suitable for hepatocyte differentiation. IHCC organoids after hepatocyte differentiation acquired functions of mature hepatocytes such as albumin secretion, bile acid production and increased CYP3A4 activity. Studies using a mouse model of IHCC indicate that Wnt3a derived from macrophages recruited upon inflammation in the liver may promote the malignant transformation of hepatocytes to IHCC cells. The results of the present study support the recently proposed hypothesis that IHCC cells are derived from hepatocytes.

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Divergent Routes toward Wnt and R-spondin Niche Independency during Human Gastric Carcinogenesis

Nanki K., Toshimitsu K., Takano A., Fujii M., Shimokawa M., Ohta Y., Matano M., Seino T., Nishikori S., Ishikawa K., Kawasaki K., Togasaki K., Takahashi S., Sukawa Y., Ishida H., Sugimoto S., Kawakubo H., Kim J., Kitagawa Y., Sekine S., Koo B., Kanai T., Sato T.

Cell （Cell）  174 （ 4 ） 856 - 869.e17 2018年08月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  0092-8674

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© 2018 Elsevier Inc. Recent sequencing analyses have shed light on heterogeneous patterns of genomic aberrations in human gastric cancers (GCs). To explore how individual genetic events translate into cancer phenotypes, we established a biological library consisting of genetically engineered gastric organoids carrying various GC mutations and 37 patient-derived organoid lines, including rare genomically stable GCs. Phenotype analyses of GC organoids revealed divergent genetic and epigenetic routes to gain Wnt and R-spondin niche independency. An unbiased phenotype-based genetic screening identified a significant association between CDH1/TP53 compound mutations and the R-spondin independency that was functionally validated by CRISPR-based knockout. Xenografting of GC organoids further established the feasibility of Wnt-targeting therapy for Wnt-dependent GCs. Our results collectively demonstrate that multifaceted genetic abnormalities render human GCs independent of the stem cell niche and highlight the validity of the genotype-phenotype screening strategy in gaining deeper understanding of human cancers. Generation and analysis of a human gastric cancer organoid bank reveal insights into molecular features underlying diverse histopathological subtypes and tumor independence from Wnt signals.

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Expansion of Adult Human Pancreatic Tissue Yields Organoids Harboring Progenitor Cells with Endocrine Differentiation Potential

Loomans C., Williams Giuliani N., Balak J., Ringnalda F., van Gurp L., Huch M., Boj S., Sato T., Kester L., de Sousa Lopes S., Roost M., Bonner-Weir S., Engelse M., Rabelink T., Heimberg H., Vries R., van Oudenaarden A., Carlotti F., Clevers H., de Koning E.

Stem Cell Reports （Stem Cell Reports）  10 （ 3 ） 1088 - 1101 2018年03月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  2213-6711

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© 2018 The Authors Generating an unlimited source of human insulin-producing cells is a prerequisite to advance β cell replacement therapy for diabetes. Here, we describe a 3D culture system that supports the expansion of adult human pancreatic tissue and the generation of a cell subpopulation with progenitor characteristics. These cells display high aldehyde dehydrogenase activity (ALDH hi ), express pancreatic progenitors markers (PDX1, PTF1A, CPA1, and MYC), and can form new organoids in contrast to ALDH lo cells. Interestingly, gene expression profiling revealed that ALDH hi cells are closer to human fetal pancreatic tissue compared with adult pancreatic tissue. Endocrine lineage markers were detected upon in vitro differentiation. Engrafted organoids differentiated toward insulin-positive (INS + ) cells, and circulating human C-peptide was detected upon glucose challenge 1 month after transplantation. Engrafted ALDH hi cells formed INS + cells. We conclude that adult human pancreatic tissue has potential for expansion into 3D structures harboring progenitor cells with endocrine differentiation potential. In the context of β cell replacement therapy for diabetes, de Koning and colleagues describe a 3D culture platform that supports ex vivo expansion of human pancreatic tissue as organoids. These organoids harbor a subpopulation of ALDH hi cells that display proliferative capacity and can differentiate to an endocrine fate.

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Gastroenteropancreatic neuroendocrine neoplasms: genes, therapies and models

Kawasaki K., Fujii M., Sato T.

Disease models & mechanisms （Disease models & mechanisms）  11 （ 2 ）  2018年02月

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© 2018. Published by The Company of Biologists Ltd. Gastroenteropancreatic neuroendocrine neoplasms (GEP-NENs) refer to a group of heterogeneous cancers of neuroendocrine cell phenotype that mainly fall into one of two subtypes: gastroenteropancreatic neuroendocrine tumors (GEP-NETs; well differentiated) or gastroenteropancreatic neuroendocrine carcinomas (GEP-NECs; poorly differentiated). Although originally defined as orphan cancers, their steadily increasing incidence highlights the need to better understand their etiology. Accumulating epidemiological and clinical data have shed light on the pathological characteristics of these diseases. However, the relatively low number of patients has hampered conducting large-scale clinical trials and hence the development of novel treatment strategies. To overcome this limitation, tractable disease models that faithfully reflect clinical features of these diseases are needed. In this Review, we summarize the current understanding of the genetics and biology of these diseases based on conventional disease models, such as genetically engineered mouse models (GEMMs) and cell lines, and discuss the phenotypic differences between the models and affected humans. We also highlight the emerging disease models derived from human clinical samples, including patient-derived xenograft models and organoids, which may provide biological and therapeutic insights into GEP-NENs.

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Reconstruction of the Human Colon Epithelium In Vivo

Sugimoto S., Ohta Y., Fujii M., Matano M., Shimokawa M., Nanki K., Date S., Nishikori S., Nakazato Y., Nakamura T., Kanai T., Sato T.

Cell Stem Cell （Cell Stem Cell）  22 （ 2 ） 171 - 176.e5 2018年02月

研究論文（学術雑誌）, 共著,  ISSN  1934-5909

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© 2017 Elsevier Inc. Genetic lineage tracing has revealed that Lgr5 + murine colon stem cells (CoSCs) rapidly proliferate at the crypt bottom. However, the spatiotemporal dynamics of human CoSCs in vivo have remained experimentally intractable. Here we established an orthotopic xenograft system for normal human colon organoids, enabling stable reconstruction of the human colon epithelium in vivo. Xenografted organoids were prone to displacement by the remaining murine crypts, and this could be overcome by complete removal of the mouse epithelium. Xenografted organoids formed crypt structures distinctively different from surrounding mouse crypts, reflecting their human origin. Lineage tracing using CRISPR-Cas9 to engineer an LGR5-CreER knockin allele demonstrated self-renewal and multipotency of LGR5 + CoSCs. In contrast to the rapidly cycling properties of mouse Lgr5 + CoSCs, human LGR5 + CoSCs were slow-cycling in vivo. This organoid-based orthotopic xenograft model enables investigation of the functional behaviors of human CoSCs in vivo, with potential therapeutic applications in regenerative medicine. Sugimoto et al. established an orthotopic xenotransplantation system for human normal colon organoids. Using LGR5-CreER knockin organoids for genetic lineage tracing, they demonstrated the self-renewal and multipotency of LGR5 + colon stem cells in mouse colon. Interestingly, human LGR5 + colon stem cells showed slower cycling than those of mice in vivo.

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Development of intestinal M cells and follicle-associated epithelium is regulated by TRAF6-mediated NF-κB signaling

Kanaya T., Sakakibara S., Jinnohara T., Hachisuka M., Tachibana N., Hidano S., Kobayashi T., Kimura S., Iwanaga T., Nakagawa T., Katsuno T., Kato N., Akiyama T., Sato T., Williams I., Ohno H.

Journal of Experimental Medicine （Journal of Experimental Medicine）  215 （ 2 ） 501 - 519 2018年02月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  0022-1007

　概要を見る

© 2018 Kanaya et al. M cells are located in the follicle-associated epithelium (FAE) that covers Peyer's patches (PPs) and are responsible for the uptake of intestinal antigens. The differentiation of M cells is initiated by receptor activator of NF-κB. However, the intracellular pathways involved in M cell differentiation are still elusive. In this study, we demonstrate that the NF-κB pathway activated by RANK is essential for M cell differentiation using in vitro organoid culture. Overexpression of NF-κB transcription factors enhances the expression of M cell-associated molecules but is not sufficient to complete M cell differentiation. Furthermore, we evaluated the requirement for tumor necrosis factor receptor-associated factor 6 (TRAF6). Conditional deletion of TRAF6 in the intestinal epithelium causes a complete loss of M cells in PPs, resulting in impaired antigen uptake into PPs. In addition, the expression of FAE-associated genes is almost silenced in TRAF6-deficient mice. This study thus demonstrates the crucial role of TRAF6-mediated NF-κB signaling in the development of M cells and FAE.

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Lectin ZG16p inhibits proliferation of human colorectal cancer cells via its carbohydrate-binding sites

Mito A., Nakano Y., Saitoh T., Gouraud S., Yamaguchi Y., Sato T., Sasaki N., Kojima-Aikawa K.

Glycobiology （Glycobiology）  28 （ 1 ） 21 - 31 2018年01月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  0959-6658

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© The Author 2017. Zymogen granule protein 16 (ZG16p) is a soluble lectin that binds to both mannose and heparin/ heparan sulfate. It is highly expressed in the human digestive tract and is secreted into the mucus. In this study, we investigated the effect of ZG16p on the proliferation of human colorectal cancer cells. Overexpression of ZG16p in Caco-2 cells decreased cell growth. Recombinant ZG16p markedly inhibited proliferation of Caco-2, LS174T, HCT116 and HCT15 cells. Caco-2 cell growth was not inhibited by two mutated ZG16p proteins, D151A and M5 (K36A, R37A, R53A, R55A and R79A) lacking mannose- and heparin-binding activities, respectively. Immunofluorescent cell staining revealed that ZG16p-D151A maintained its binding to the Caco-2 cell surface, whereas ZG16p-M5 failed to bind to the cells. These results suggest that ZG16p interacts with the cell surface via basic amino acids substituted in ZG16p-M5 and inhibits Caco-2 cell proliferation via Asp151. In addition, growth of patient-derived colorectal tumor organoids in a 3D intestinal stem cell system was suppressed by ZG16p but not by ZG16p-M5. Taken together, our findings indicate that ZG16p inhibits the growth of colorectal cancer cells via its carbohydrate-binding sites in vitro and ex vivo. In this study, a novel pathway in cancer cell growth regulation through cell surface carbohydrate chains is suggested.

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Advancing Intestinal Organoid Technology Toward Regenerative Medicine

Nakamura T., Sato T.

Cellular and Molecular Gastroenterology and Hepatology （Cellular and Molecular Gastroenterology and Hepatology）  5 （ 1 ） 51 - 60 2018年01月

研究論文（学術雑誌）, 共著, 査読有り

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© 2018 The Authors With the emergence of technologies to culture intestinal epithelial cells in vitro as various forms of intestinal organoids, there is growing interest in using such cultured intestinal cells as a source for tissue engineering and regenerative medicine. One such approach would be to combine the organoid technology with methodologies to engineer the culture environment, particularly the three-dimensional scaffold materials, to generate intestines that exquisitely recapitulate their original structures and functions. Another approach to use organoids for regenerative medicine would be to urge them to mature into functional intestines by implanting them into hosts. This process includes the tissue-engineered small intestine that uses synthetic scaffolds for tissue regeneration and direct organoid transplantation that takes advantage of submucosal tissues in the native intestines as a scaffold. Further study in these subfields could lead to the development of therapeutic options to use different types of organoids with various cell types in regenerative medicine for intestinal diseases in humans.

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Human Pancreatic Tumor Organoids Reveal Loss of Stem Cell Niche Factor Dependence during Disease Progression

Seino T., Kawasaki S., Shimokawa M., Tamagawa H., Toshimitsu K., Fujii M., Ohta Y., Matano M., Nanki K., Kawasaki K., Takahashi S., Sugimoto S., Iwasaki E., Takagi J., Itoi T., Kitago M., Kitagawa Y., Kanai T., Sato T.

Cell Stem Cell （Cell Stem Cell）  22 （ 3 ） 454 - 467 2018年01月

研究論文（学術雑誌）, 査読有り,  ISSN  1934-5909

　概要を見る

© 2017 Elsevier Inc. Despite recent efforts to dissect the inter-tumor heterogeneity of pancreatic ductal adenocarcinoma (PDAC) by determining prognosis-predictive gene expression signatures for specific subtypes, their functional differences remain elusive. Here, we established a pancreatic tumor organoid library encompassing 39 patient-derived PDACs and identified 3 functional subtypes based on their stem cell niche factor dependencies on Wnt and R-spondin. A Wnt-non-producing subtype required Wnt from cancer-associated fibroblasts, whereas a Wnt-producing subtype autonomously secreted Wnt ligands and an R-spondin-independent subtype grew in the absence of Wnt and R-spondin. Transcriptome analysis of PDAC organoids revealed gene-expression signatures that associated Wnt niche subtypes with GATA6-dependent gene expression subtypes, which were functionally supported by genetic perturbation of GATA6. Furthermore, CRISPR-Cas9-based genome editing of PDAC driver genes (KRAS, CDKN2A, SMAD4, and TP53) demonstrated non-genetic acquisition of Wnt niche independence during pancreas tumorigenesis. Collectively, our results reveal functional heterogeneity of Wnt niche independency in PDAC that is non-genetically formed through tumor progression. Sato and colleagues established a library of patient-derived pancreas cancer organoids and identified heterogeneous patterns of dependency on Wnt ligands among pancreas cancers. Biological and genetic analyses highlighted GATA6 as a mediator of the Wnt niche requirement, which links pancreatic tumor progression to independence from the stem cell niche.

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Intestinal Dysbiosis and Biotin Deprivation Induce Alopecia through Overgrowth of Lactobacillus murinus in Mice

Hayashi Atsushi, Mikami Yohei, Miyamoto Kentaro, Kamada Nobuhiko, Sato Toshiro, Mizuno Shinta, Naganuma Makoto, Teratani Toshiaki, Aoki Ryo, Fukuda Shinji, Suda Wataru, Hattori Masahira, Amagai Masayuki, Ohyama Manabu, Kanai Takanori

Cell Reports 20 （ 7 ） 1513 - 1524 2017年08月

研究論文（学術雑誌）, 共著,  ISSN  2211-1247

　概要を見る

Metabolism by the gut microbiota affects host physiology beyond the gastrointestinal tract. Here, we find that antibiotic-induced dysbiosis, in particular, overgrowth of Lactobacillus murinus (L. murinus), impaired gut metabolic function and led to the development of alopecia. While deprivation of dietary biotin per se did not affect skin physiology, its simultaneous treatment with vancomycin resulted in hair loss in specific pathogen-free (SPF) mice. Vancomycin treatment induced the accumulation of L. murinus in the gut, which consumes residual biotin and depletes available biotin in the gut. Consistently, L. murinus induced alopecia when monocolonized in germ-free mice fed a biotin-deficient diet. Supplementation of biotin can reverse established alopecia symptoms in the SPF condition, indicating that L. murinus plays a central role in the induction of hair loss via a biotin-dependent manner. Collectively, our results indicate that luminal metabolic alterations associated with gut dysbiosis and dietary modifications can compromise skin physiology.

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Defining the role of Lgr5⁺ stem cells in colorectal cancer

Fujii Masayuki, Sato Toshiro

Genome Medicine 9 （ 1 ）  2017年07月

研究論文（学術雑誌）, 共著,  ISSN  1756-994X

　概要を見る

Intestinal epithelium is structured by two distinct components: the villi and the crypts. The crypts harbor stem cells expressing Lgr5 and thus have been a representative model to study tissue stem cell functions. Recent advances in organoid technology and analytical modalities have enabled precise characterization of Lgr5⁺ intestinal stem cells, providing insights into their roles in homeostasis and cancer.

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Establishment of 3D intestinal organoid cultures from intestinal stem cells

杉本 真也, 佐藤 俊朗

Methods in Molecular Biology （Springer）  1612   97 - 105 2017年06月

研究論文（学術雑誌）, 共著, 査読有り

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<p>The intestinal epithelium is the most rapidly renewed tissue in adult mammals, and its renewal is strictly controlled by intestinal stem cells. Extensive studies using genetic models of intestinal epithelium have revealed the mechanisms underlying the self-renewal of intestinal stem cells. Exploiting this knowledge, we developed a novel 3D culture system that enables the outgrowth of intestinal Lgr5⁺ stem cells derived from mouse and human tissues into ever-expanding crypt–villus mini-guts, known as intestinal epithelial organoids. These organoids are maintained by the self-renewal of stem cells and give rise to all differentiated cell types of the intestinal epithelium. Once established, organoids can be cryopreserved and thawed when needed. This culture system has been widely used for studying stem cell behavior and gene function and for disease modeling.</p>

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IL-22BP dictates characteristics of Peyer's patch follicleassociated epithelium for antigen uptake

Jinnohara Toshi, Kanaya Takashi, Hase Koji, Sakakibara Sayuri, Kato Tamotsu, Tachibana Naoko, Sasaki Takaharu, Hashimoto Yusuke, Sato Toshiro, Watarai Hiroshi, Kunisawa Jun, Shibata Naoko, Williams Ifor R., Kiyono Hiroshi, Ohno Hiroshi

Journal of Experimental Medicine 214 （ 6 ） 1607 - 1618 2017年06月

研究論文（学術雑誌）, 共著,  ISSN  0022-1007

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Interleukin-22 (IL-22) acts protectively and harmfully on intestinal tissue depending on the situation; therefore, IL-22 signaling needs to be tightly regulated. IL-22 binding protein (IL-22BP) binds IL-22 to inhibit IL-22 signaling. It is expressed in intestinal and lymphoid tissues, although its precise distribution and roles have remained unclear. In this study, we show that IL-22BP is highly expressed by CD11b⁺CD8α- dendritic cells in the subepithelial dome region of Peyer's patches (PPs). We found that IL-22BP blocks IL-22 signaling in the follicle-associated epithelium (FAE) covering PPs, indicating that IL-22BP plays a role in regulating the characteristics of the FAE. As expected, FAE of IL-22BP-deficient (Il22ra2^-/-) mice exhibited altered properties such as the enhanced expression of mucus and antimicrobial proteins as well as prominent fucosylation, which are normally suppressed in FAE. Additionally, Il22ra2^-/- mice exhibited the decreased uptake of bacterial antigens into PPs without affecting M cell function. Our present study thus demonstrates that IL-22BP promotes bacterial uptake into PPs by influencing FAE gene expression and function.

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Visualization and targeting of LGR5 + human colon cancer stem cells

Shimokawa Mariko, Ohta Yuki, Nishikori Shingo, Matano Mami, Takano Ai, Fujii Masayuki, Date Shoichi, Sugimoto Shinya, Kanai Takanori, Sato Toshiro

Nature 545 （ 7653 ） 187 - 192 2017年05月

研究論文（学術雑誌）, 共著,  ISSN  0028-0836

　概要を見る

The cancer stem cell (CSC) theory highlights a self-renewing subpopulation of cancer cells that fuels tumour growth. The existence of human CSCs is mainly supported by xenotransplantation of prospectively isolated cells, but their clonal dynamics and plasticity remain unclear. Here, we show that human LGR5 + colorectal cancer cells serve as CSCs in growing cancer tissues. Lineage-tracing experiments with a tamoxifen-inducible Cre knock-in allele of LGR5 reveal the self-renewal and differentiation capacity of LGR5 + tumour cells. Selective ablation of LGR5 + CSCs in LGR5-iCaspase9 knock-in organoids leads to tumour regression, followed by tumour regrowth driven by re-emerging LGR5 + CSCs. KRT20 knock-in reporter marks differentiated cancer cells that constantly diminish in tumour tissues, while reverting to LGR5 + CSCs and contributing to tumour regrowth after LGR5 + CSC ablation. We also show that combined chemotherapy potentiates targeting of LGR5 + CSCs. These data provide insights into the plasticity of CSCs and their potential as a therapeutic target in human colorectal cancer.

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Cell competition with normal epithelial cells promotes apical extrusion of transformed cells through metabolic changes

Kon Shunsuke, Ishibashi Kojiro, Katoh Hiroto, Kitamoto Sho, Shirai Takanobu, Tanaka Shinya, Kajita Mihoko, Ishikawa Susumu, Yamauchi Hajime, Yako Yuta, Kamasaki Tomoko, Matsumoto Tomohiro, Watanabe Hirotaka, Egami Riku, Sasaki Ayana, Nishikawa Atsuko, Kameda Ikumi, Maruyama Takeshi, Narumi Rika, Morita Tomoko, Sasaki Yoshiteru, Enoki Ryosuke, Honma Sato, Imamura Hiromi, Oshima Masanobu, Soga Tomoyoshi, Miyazaki Jun Ichi, Duchen Michael R., Nam Jin Min, Onodera Yasuhito, Yoshioka Shingo, Kikuta Junichi, Ishii Masaru, Imajo Masamichi, Nishida Eisuke, Fujioka Yoichiro, Ohba Yusuke, Sato Toshiro, Fujita Yasuyuki

Nature Cell Biology 19 （ 5 ） 530 - 541 2017年05月

研究論文（学術雑誌）, 共著,  ISSN  1465-7392

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Recent studies have revealed that newly emerging transformed cells are often apically extruded from epithelial tissues. During this process, normal epithelial cells can recognize and actively eliminate transformed cells, a process called epithelial defence against cancer (EDAC). Here, we show that mitochondrial membrane potential is diminished in RasV12-transformed cells when they are surrounded by normal cells. In addition, glucose uptake is elevated, leading to higher lactate production. The mitochondrial dysfunction is driven by upregulation of pyruvate dehydrogenase kinase 4 (PDK4), which positively regulates elimination of RasV12-transformed cells. Furthermore, EDAC from the surrounding normal cells, involving filamin, drives the Warburg-effect-like metabolic alteration. Moreover, using a cell-competition mouse model, we demonstrate that PDK-mediated metabolic changes promote the elimination of RasV12-transformed cells from intestinal epithelia. These data indicate that non-cell-autonomous metabolic modulation is a crucial regulator for cell competition, shedding light on the unexplored events at the initial stage of carcinogenesis.

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Intestinal Epithelial Lgr5⁺ Stem Cell Niche and Organoids

Sasaki Nobuo, Sato Toshiro, Clevers Hans

Biology and Engineering of Stem Cell Niches （Elsevier）     111 - 125 2017年04月

共著

　概要を見る

<p>Over the past 10years, insights into the biology of intestinal stem cells have rapidly increased. The adult intestinal epithelium represents one of the most suitable models to study tissue stem cells. It is the rapidly self-renewing tissue and the design of its basic units, the crypt-villus is highly stereotypical. Since the discovery of Lgr5⁺ stem cells at the base of intestinal crypts, it has become possible to unveil the molecular mechanisms that control homeostasis of the stem cells, including the extrinsic signaling cues from the crypt niche. Based on these insights, in vitro propagation of murine and human intestinal stem cells in the form of ever-expanding organoids has become possible. The three-dimensional culture technology of organoids opens new avenues for studying fundamental questions on the stem cells and their niche, but has also allowed several applications in personalized medicine. Here, we discuss these recent developments and insights.</p>

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Zinc Transporter SLC39A7/ZIP7 Promotes Intestinal Epithelial Self-Renewal by Resolving ER Stress

Ohashi Wakana, Kimura Shunsuke, Iwanaga Toshihiko, Furusawa Yukihiro, Irié Tarou, Izumi Hironori, Watanabe Takashi, Hijikata Atsushi, Hara Takafumi, Ohara Osamu, Koseki Haruhiko, Sato Toshiro, Robine Sylvie, Mori Hisashi, Hattori Yuichi, Watarai Hiroshi, Mishima Kenji, Ohno Hiroshi, Hase Koji, Fukada Toshiyuki

PLoS Genetics 12 （ 10 ）  2016年10月

研究論文（学術雑誌）,  ISSN  1553-7390

　概要を見る

Zinc transporters play a critical role in spatiotemporal regulation of zinc homeostasis. Although disruption of zinc homeostasis has been implicated in disorders such as intestinal inflammation and aberrant epithelial morphology, it is largely unknown which zinc transporters are responsible for the intestinal epithelial homeostasis. Here, we show that Zrt-Irt-like protein (ZIP) transporter ZIP7, which is highly expressed in the intestinal crypt, is essential for intestinal epithelial proliferation. Mice lacking Zip7 in intestinal epithelium triggered endoplasmic reticulum (ER) stress in proliferative progenitor cells, leading to significant cell death of progenitor cells. Zip7 deficiency led to the loss of Olfm4⁺ intestinal stem cells and the degeneration of post-mitotic Paneth cells, indicating a fundamental requirement for Zip7 in homeostatic intestinal regeneration. Taken together, these findings provide evidence for the importance of ZIP7 in maintenance of intestinal epithelial homeostasis through the regulation of ER function in proliferative progenitor cells and maintenance of intestinal stem cells. Therapeutic targeting of ZIP7 could lead to effective treatment of gastrointestinal disorders.

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Dephosphorylated parafibromin is a transcriptional coactivator of the Wnt/Hedgehog/Notch pathways

Kikuchi Ippei, Takahashi-Kanemitsu Atsushi, Sakiyama Natsuki, Tang Chao, Tang Pei Jung, Noda Saori, Nakao Kazuki, Kassai Hidetoshi, Sato Toshiro, Aiba Atsu, Hatakeyama Masanori

Nature Communications 7 2016年09月

研究論文（学術雑誌）, 共著,  ISSN  2041-1723

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Evolutionally conserved Wnt, Hedgehog (Hh) and Notch morphogen pathways play essential roles in the development, homeostasis and pathogenesis of multicellular organisms. Nevertheless, mechanisms that intracellularly coordinate these signal inputs remain poorly understood. Here we found that parafibromin, a component of the PAF complex, competitively interacts with β-catenin and Gli1, thereby potentiating transactivation of Wnt- and Hh-target genes in a mutually exclusive manner. Parafibromin also binds to the Notch intracellular domain (NICD), enabling concerted activation of Wnt- and Notch-target genes. The transcriptional platform function of parafibromin is potentiated by tyrosine dephosphorylation, mediated by SHP2 phosphatase, while it is attenuated by tyrosine phosphorylation, mediated by PTK6 kinase. Consequently, acute loss of parafibromin in mice disorganizes the normal epithelial architecture of the intestine, which requires coordinated activation/inactivation of Wnt, Hh and/or Notch signalling. Parafibromin integrates and converts signals conveyed by these morphogen pathways into appropriate transcriptional outputs in a tyrosine phosphorylation/dephosphorylation-regulated manner.

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Mule Regulates the Intestinal Stem Cell Niche via the Wnt Pathway and Targets EphB3 for Proteasomal and Lysosomal Degradation

Dominguez-Brauer Carmen, Hao Zhenyue, Elia Andrew J., Fortin Jérôme M., Nechanitzky Robert, Brauer Patrick M., Sheng Yi, Mana Miyeko D., Chio Iok In Christine, Haight Jillian, Pollett Aaron, Cairns Robert, Tworzyanski Leanne, Inoue Satoshi, Reardon Colin, Marques Ana, Silvester Jennifer, Cox Maureen A., Wakeham Andrew, Yilmaz Omer H., Sabatini David M., van Es Johan H., Clevers Hans, Sato Toshiro, Mak Tak W.

Cell Stem Cell 19 （ 2 ） 205 - 216 2016年08月

研究論文（学術雑誌）, 共著,  ISSN  1934-5909

　概要を見る

The E3 ubiquitin ligase Mule is often overexpressed in human colorectal cancers, but its role in gut tumorigenesis is unknown. Here, we show in vivo that Mule controls murine intestinal stem and progenitor cell proliferation by modulating Wnt signaling via c-Myc. Mule also regulates protein levels of the receptor tyrosine kinase EphB3 by targeting it for proteasomal and lysosomal degradation. In the intestine, EphB/ephrinB interactions position cells along the crypt-villus axis and compartmentalize incipient colorectal tumors. Our study thus unveils an important new avenue by which Mule acts as an intestinal tumor suppressor by regulation of the intestinal stem cell niche.

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A Colorectal Tumor Organoid Library Demonstrates Progressive Loss of Niche Factor Requirements during Tumorigenesis

Fujii Masayuki, Shimokawa Mariko, Date Shoichi, Takano Ai, Matano Mami, Nanki Kosaku, Ohta Yuki, Toshimitsu Kohta, Nakazato Yoshihiro, Kawasaki Kenta, Uraoka Toshio, Watanabe Toshiaki, Kanai Takanori, Sato Toshiro

Cell Stem Cell 18 （ 6 ） 827 - 838 2016年06月

研究論文（学術雑誌）, 共著,  ISSN  1934-5909

　概要を見る

Summary Colorectal tumor is a heterogeneous disease, with varying clinical presentation and prognosis in patients. To establish a platform encompassing this diversity, we generated 55 colorectal tumor organoid lines from a range of histological subtypes and clinical stages, including rare subtypes. Each line was defined by gene expression signatures and optimized for organoid culture according to niche factor requirements. In vitro and in xenografts, the organoids reproduced the histopathological grade and differentiation capacity of their parental tumors. Notably, we found that niche-independent growth is predominantly associated with the adenoma-carcinoma transition reflecting accumulation of multiple mutations. For matched pairs of primary and metastatic organoids, which had similar genetic profiles and niche factor requirements, the metastasis-derived organoids exhibited higher metastatic capacity. These observations underscore the importance of genotype-phenotype analyses at a single-patient level and the value of our resource to provide insights into colorectal tumorigenesis and patient-centered therapeutic development.

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Inhibition of DNA Methylation Suppresses Intestinal Tumor Organoids by Inducing an Anti-Viral Response

Saito Yoshimasa, Nakaoka Toshiaki, Sakai Kasumi, Muramatsu Toshihide, Toshimitsu Kohta, Kimura Masaki, Kanai Takanori, Sato Toshiro, Saito Hidetsugu

Scientific Reports 6 2016年05月

共著

　概要を見る

Recent studies have proposed that the major anti-tumor effect of DNA methylation inhibitors is induction of interferon-responsive genes via dsRNAs-containing endogenous retroviruses. Recently, a 3D culture system for stem cells known as organoid culture has been developed. Lgr5-positive stem cells form organoids that closely recapitulate the properties of original tissues. To investigate the effect of DNA demethylation on tumor organoids, we have established organoids from intestinal tumors of Apc^Min/+ (Min) mice and subjected them to 5-aza-2′-deoxycytidine (5-Aza-CdR) treatment and Dnmt1 knockdown. DNA demethylation induced by 5-Aza-CdR treatment and Dnmt1 knockdown significantly reduced the cell proliferation of the tumor organoids. Microarray analyses of the tumor organoids after 5-Aza-CdR treatment and Dnmt1 knockdown revealed that interferon-responsive genes were activated by DNA demethylation. Gene ontology and pathway analyses clearly demonstrated that these genes activated by DNA demethylation are involved in the anti-viral response. These findings indicate that DNA demethylation suppresses the proliferation of intestinal tumor organoids by inducing an anti-viral response including activation of interferon-responsive genes. Treatment with DNA methylation inhibitors to activate a growth-inhibiting immune response may be an effective therapeutic approach for colon cancers.

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SETD7 Controls Intestinal Regeneration and Tumorigenesis by Regulating Wnt/β-Catenin and Hippo/YAP Signaling

Oudhoff Menno J., Braam Mitchell J S, Freeman Spencer A., Wong Denise, Rattray David G., Wang Jia, Antignano Frann, Snyder Kimberly, Refaeli Ido, Hughes Michael R., McNagny Kelly M., Gold Michael R., Arrowsmith Cheryl H., Sato Toshiro, Rossi Fabio M V, Tatlock John H., Owen Dafydd R., Brown Peter J., Zaph Colby

Developmental Cell 37 （ 1 ） 47 - 57 2016年04月

研究論文（学術雑誌）, 共著,  ISSN  1534-5807

　概要を見る

Intestinal tumorigenesis is a result of mutations in signaling pathways that control cellular proliferation, differentiation, and survival. Mutations in the Wnt/β-catenin pathway are associated with the majority of intestinal cancers, while dysregulation of the Hippo/Yes-Associated Protein (YAP) pathway is an emerging regulator of intestinal tumorigenesis. In addition, these closely related pathways play a central role during intestinal regeneration. We have previously shown that methylation of the Hippo transducer YAP by the lysine methyltransferase SETD7 controls its subcellular localization and function. We now show that SETD7 is required for Wnt-driven intestinal tumorigenesis and regeneration. Mechanistically, SETD7 is part of a complex containing YAP, AXIN1, and β-catenin, and SETD7-dependent methylation of YAP facilitates Wnt-induced nuclear accumulation of β-catenin. Collectively, these results define a methyltransferase-dependent regulatory mechanism that links the Wnt/β-catenin and Hippo/YAP pathways during intestinal regeneration and tumorigenesis. The Wnt/β-catenin and Hippo/YAP pathways are intimately linked in structure and function. Oudhoff et al. provide evidence that the lysine methyltransferase SETD7 is a central link between these pathways. SETD7-dependent methylation of YAP is required for optimal β-catenin-dependent gene expression. Accordingly, loss of SETD7 attenuates Wnt-dependent intestinal tumorigenesis and regeneration.

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Active and water-soluble form of lipidated wnt protein is maintained by a serum glycoprotein afamin/α-albumin

Mihara Emiko, Hirai Hidenori, Yamamoto Hideki, Tamura-Kawakami Keiko, Matano Mami, Kikuchi Akira, Sato Toshiro, Takagi Junichi

eLife 5 （ FEBRUARY2016 ）  2016年02月

研究論文（学術雑誌）, 共著,  ISSN  2050-084X

　概要を見る

Wnt plays important role during development and in various diseases. Because Wants are lipidated and highly hydrophobic, they can only be purified in the presence of detergents, limiting their use in various in vitro and in vivo assays. We purified N-terminally tagged recombinant Wnt3a secreted from cells and accidentally discovered that Wnt3a co-purified with a glycoprotein afamin derived from the bovine serum included in the media. Wnt3a forms a 1:1 complex with afamin, which remains soluble in aqueous buffer after isolation, and can induce signaling in various cellular systems including the intestinal stem cell growth assay. By co-expressing with afamin, biologically active afamin-Wnt complex can be easily obtained in large quantity. As afamin can also solubilize Wnt5a, Wnt3, and many more Wnt subtypes, ataman complexation will open a way to put various Wnt ligands and their signaling mechanisms under a thorough biochemical scrutiny that had been difficult for years.

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EHBP1L1 coordinates Rab8 and Bin1 to regulate apical-directed transport in polarized epithelial cells

Nakajo Atsuhiro, Yoshimura Shin ichiro, Togawa Hiroko, Kunii Masataka, Iwano Tomohiko, Izumi Ayaka, Noguchi Yuria, Watanabe Ayako, Goto Ayako, Sato Toshiro, Harada Akihiro

Journal of Cell Biology 212 （ 3 ） 297 - 306 2016年

研究論文（学術雑誌）, 共著,  ISSN  0021-9525

　概要を見る

The highly conserved Rab guanosine triphosphatase (GTPase) Rab8 plays a role in exocytosis toward the polarized plasma membrane in eukaryotic cells. In murine Rab8-deficient small intestine cells, apical proteins are missorted into lysosomes. In this study, we identified a novel Rab8-interacting protein complex containing an EH domain-binding protein 1-like 1 (EHBP1L1), Bin1/amphiphysin II, and dynamin. Biochemical analyses showed that EHBP1L1 directly bound to GTP-loaded Rab8 and Bin1. The spatial dependency of these complexes at the endocytic recycling compartment (ERC) was demonstrated through overexpression and knockdown experiments. EHBP1L1-or Bin1-depleted or dynamininhibited small intestine organoids significantly accumulated apical membrane proteins but not basolateral membrane proteins in lysosomes. Furthermore, in EHBP1L1-deficient mice, small intestine cells displayed truncated and sparse microvilli, suggesting that EHBP1L1 maintains the apical plasma membrane by regulating apical transport. In summary, our data demonstrate that EHBP1L1 links Rab8 and the Bin1-dynamin complex, which generates membrane curvature and excises the vesicle at the ERC for apical transport.

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Mini-Gut Organoids

Date Shoichi, Sato Toshiro

Annual Review of Cell and Developmental Biology 31   269 - 289 2015年11月

研究論文（学術雑誌）, 共著,  ISSN  1081-0706

　概要を見る

In the adult mammalian body, self-renewal of tissue stem cells is regulated by extracellular niche environments in response to the demands of tissue organization. Intestinal stem cells expressing Lgr5 constantly self-renew in their specific niche at the crypt bottom to maintain rapid turnover of the epithelium. Niche-regulated stem cell self-renewal is perturbed in several mouse genetic models and during human tumorigenesis, suggesting roles for EGF, Wnt, BMPTGF-β, and Notch signaling. In vitro niche reconstitution capitalizing on this knowledge has enabled the growth of single intestinal stem cells into mini-gut epithelial organoids comprising Lgr5⁺ stem cells and all types of differentiated lineages. The mini-gut organoid culture platform is applicable to various types of digestive tissue epithelium from multiple species. The mechanism of self-renewal in organoids provides novel insights for organogenesis, regenerative medicine, and tumorigenesis of the digestive system.

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Efficient genetic engineering of human intestinal organoids using electroporation

Fujii Masayuki, Matano Mami, Nanki Kosaku, Sato Toshiro

Nature Protocols 10 （ 10 ） 1474 - 1485 2015年10月

研究論文（学術雑誌）, 共著,  ISSN  1754-2189

　概要を見る

Gene modification in untransformed human intestinal cells is an attractive approach for studying gene function in intestinal diseases. However, because of the lack of practical tools, such studies have largely depended upon surrogates, such as gene-engineered mice or immortalized human cell lines. By taking advantage of the recently developed intestinal organoid culture method, we developed a methodology for modulating genes of interest in untransformed human colonic organoids via electroporation of gene vectors. Here we describe a detailed protocol for the generation of intestinal organoids by culture with essential growth factors in a basement membrane matrix. We also describe how to stably integrate genes via the piggyBac transposon, as well as precise genome editing using the CRISPR-Cas9 system. Beginning with crypt isolation from a human colon sample, genetically modified organoids can be obtained in 3 weeks.

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SnapShot: Growing Organoids from Stem Cells.

Sato Toshiro, Clevers Hans

Cell （Elsevie）  161 （ 7 ） 1700 - 1700.e1 2015年06月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  0092-8674

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Ink4a/arf-dependent loss of parietal cells induced by oxidative stress promotes CD44-dependent gastric tumorigenesis

Seishima Ryo, Wada Takeyuki, Tsuchihashi Kenji, Okazaki Shogo, Yoshikawa Momoko, Oshima Hiroko, Oshima Masanobu, Sato Toshiro, Hasegawa Hirotoshi, Kitagawa Yuko, Goldenring James R., Saya Hideyuki, Nagano Osamu

Cancer Prevention Research 8 （ 6 ） 492 - 501 2015年06月

研究論文（学術雑誌）, 共著,  ISSN  1940-6207

　概要を見る

Loss of parietal cells initiates the development of spasmolytic polypeptide-expressing metaplasia (SPEM), a precancerous lesion in stomach. CD44 variant (CD44v) that enhances the ability to defend against reactive oxygen species (ROS) in epithelial cells is expressed de novo in SPEM of K19-Wnt1/C2mE mice, a transgenic model of gastric tumorigenesis, and is required for the efficient development of SPEM and gastric tumor in these animals. The role of ROS and its downstream signaling in CD44-dependent gastric tumorigenesis has remained unknown, however. With the use of the K19-Wnt1/C2mE mouse, we now show that parietal cells in the inflamed stomach are highly sensitive to oxidative stress and manifest activation of p38&lt;sup&gt;MAPK&lt;/sup&gt; signaling by ROS. Oral treatment with the antioxidant ascorbic acid or genetic ablation of the Ink4a/Arf locus, a major downstream target of ROS-p38&lt;sup&gt;MAPK&lt;/sup&gt; signaling, inhibited parietal cell loss and the subsequent gastric tumorigenesis. Our results indicate that signaling activated by oxidative stress in parietal cells plays a key role in CD44-dependent gastric tumorigenesis.

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Combination therapy with infliximab and thiopurine compared to infliximab monotherapy in maintaining remission of postoperative crohn's disease

Sakuraba Atsushi, Okamoto Susumu, Matsuoka Katsuyoshi, Sato Toshiro, Naganuma Makoto, Hisamatsu Tadakazu, Iwao Yasushi, Ogata Haruhiko, Kanai Takanori, Hibi Toshifumi

Digestion 91 （ 3 ） 233 - 238 2015年05月

研究論文（学術雑誌）, 共著,  ISSN  0012-2823

　概要を見る

Background and Aims: Infliximab is an efficacious agent used for the induction and maintenance of remission in Crohn's disease (CD), and recent studies suggested that it may also prevent the recurrence of this disease after surgery. The present study was performed to assess the efficacy and safety of infliximab in the postoperative setting, and to identify whether combination treatment with thiopurines had any additional beneficial effect as compared to mono-therapy. Methods: We performed a retrospective cohort study to compare the efficacy of infliximab mono-therapy and combination treatment with a thiopurine in preventing recurrence after surgery. Results: Forty-one patients who received infliximab as maintenance treatment following surgery from May 2002 to April 2010 were identified. Twenty-four were naive to infliximab, and 17 who underwent surgery during infliximab treatment were continued on it following surgery. The median follow-up period was 27 months (range 12-66 months). All patients continued infliximab as maintenance treatment, but 10 required dose intensification due to clinical recurrence. Kaplan-Meier analysis demonstrated that the use of concomitant thiopurine was correlated with the continuation of infliximab treatment at an 8-week interval (log-rank test p = 0.018). The rate of adverse event was 9.8% with no patient experiencing severe adverse reactions. Conclusion: Infliximab appears to be safe and it prevented clinical recurrence after surgery. Concomitant thiopurine use predicted response toward continuation of therapy at an 8-week interval. Prospective controlled studies to assess the efficacy of combination treatment in the postoperative setting are warranted.

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Suppressing TGFβ signaling in regenerating epithelia in an inflammatory microenvironment is sufficient to cause invasive intestinal cancer

Oshima Hiroko, Nakayama Mizuho, Han Tae Su, Naoi Kuniko, Ju Xiaoli, Maeda Yusuke, Robine Sylvie, Tsuchiya Kiichiro, Sato Toshiro, Sato Hiroshi, Taketo Makoto Mark, Oshima Masanobu

Cancer Research 75 （ 4 ） 766 - 776 2015年02月

研究論文（学術雑誌）, 共著,  ISSN  0008-5472

　概要を見る

Genetic alterations in the TGFβ signaling pathway in combination with oncogenic alterations lead to cancer development in the intestines. However, the mechanisms of TGFβ signaling suppression in malignant progression of intestinal tumors have not yet been fully understood. We have examined Apc^Δ716 TGFβr2ΔIEC compound mutant mice that carry mutations in Apc and TGFβr2 genes in the intestinal epithelial cells. We found inflammatory microenvironment only in the invasive intestinal adenocarcinomas but not in noninvasive benign polyps of the same mice. We thus treated simple TGFβr2ΔIEC mice with dextran sodium sulfate (DSS) that causes ulcerative colitis. Importantly, these TGFβr2ΔIEC mice developed invasive colon cancer associated with chronic inflammation.Wealso found that TGFβ signaling is suppressed in human colitis-associated colon cancer cells. In the mouse invasive tumors, macrophages infiltrated and expressed MT1-MMP, causingMMP2activation. These results suggest that inflammatory microenvironment contributes to submucosal invasion of TGFβ signaling-repressed epithelial cells through activation of MMP2. We further found that regeneration was impaired in TGFβr2ΔIEC mice for intestinal mucosa damaged by DSS treatment or X-ray irradiation, resulting in the expansion of undifferentiated epithelial cell population. Moreover, organoids of intestinal epithelial cells cultured from irradiated TGFβr2ΔIEC mice formed "long crypts" in Matrigel, suggesting acquisition of an invasive phenotype into the extracellular matrix. These results, taken together, indicate that a simple genetic alteration in the TGFβ signaling pathway in the inflamed and regenerating intestinal mucosa can cause invasive intestinal tumors. Such a mechanism may play a role in the colon carcinogenesis associated with inflammatory bowel disease in humans.

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An individual based computational model of intestinal crypt fission and its application to predicting unrestrictive growth of the intestinal epithelium

Pin Carmen, Parker Aimee, Gunning A. Patrick, Ohta Yuki, Johnson Ian T., Carding Simon R., Sato Toshiro

Integrative Biology (United Kingdom) 7 （ 2 ） 213 - 228 2015年02月

研究論文（学術雑誌）, 共著,  ISSN  1757-9694

　概要を見る

Intestinal crypt fission is a homeostatic phenomenon, observable in healthy adult mucosa, but which also plays a pathological role as the main mode of growth of some intestinal polyps. Building on our previous individual based model for the small intestinal crypt and on in vitro cultured intestinal organoids, we here model crypt fission as a budding process based on fluid mechanics at the individual cell level and extrapolated predictions for growth of the intestinal epithelium. Budding was always observed in regions of organoids with abundant Paneth cells. Our data support a model in which buds are biomechanically initiated by single stem cells surrounded by Paneth cells which exhibit greater resistance to viscoelastic deformation, a hypothesis supported by atomic force measurements of single cells. Time intervals between consecutive budding events, as simulated by the model and observed in vitro, were 2.84 and 2.62 days, respectively. Predicted cell dynamics was unaffected within the original crypt which retained its full capability of providing cells to the epithelium throughout fission. Mitotic pressure in simulated primary crypts forced upward migration of buds, which simultaneously grew into new protruding crypts at a rate equal to 1.03 days(-1) in simulations and 0.99 days(-1) in cultured organoids. Simulated crypts reached their final size in 4.6 days, and required 6.2 days to migrate to the top of the primary crypt. The growth of the secondary crypt is independent of its migration along the original crypt. Assuming unrestricted crypt fission and multiple budding events, a maximal growth rate of the intestinal epithelium of 0.10 days(-1) is predicted and thus approximately 22 days are required for a 10-fold increase of polyp size. These predictions are in agreement with the time reported to develop macroscopic adenomas in mice after loss of Apc in intestinal stem cells.

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Modeling colorectal cancer using CRISPR-Cas9-mediated engineering of human intestinal organoids

Matano Mami, Date Shoichi, Shimokawa Mariko, Takano Ai, Fujii Masayuki, Ohta Yuki, Watanabe Toshiaki, Kanai Takanori, Sato Toshiro

Nature Medicine 21 （ 3 ） 256 - 262 2015年

研究論文（学術雑誌）, 共著,  ISSN  1078-8956

　概要を見る

Human colorectal tumors bear recurrent mutations in genes encoding proteins operative in the WNT, MAPK, TGF-β, TP53 and PI3K pathways. Although these pathways influence intestinal stem cell niche signaling, the extent to which mutations in these pathways contribute to human colorectal carcinogenesis remains unclear. Here we use the CRISPR-Cas9 genome-editing system to introduce multiple such mutations into organoids derived from normal human intestinal epithelium. By modulating the culture conditions to mimic that of the intestinal niche, we selected isogenic organoids harboring mutations in the tumor suppressor genes APC, SMAD4 and TP53, and in the oncogenes KRAS and/or PIK3CA. Organoids engineered to express all five mutations grew independently of niche factors in vitro, and they formed tumors after implantation under the kidney subcapsule in mice. Although they formed micrometastases containing dormant tumor-initiating cells after injection into the spleen of mice, they failed to colonize in the liver. In contrast, engineered organoids derived from chromosome-instable human adenomas formed macrometastatic colonies. These results suggest that 'driver' pathway mutations enable stem cell maintenance in the hostile tumor microenvironment, but that additional molecular lesions are required for invasive behavior.

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Back to 2D Culture for Ground State of Intestinal Stem Cells

Shimokawa Mariko, Sato Toshiro

Cell Stem Cell 17 （ 1 ） 5 - 7 2015年

研究論文（学術雑誌）, 共著,  ISSN  1934-5909

　概要を見る

Generating highly uniform clonogenic stem cell populations has been remarkably difficult with stem cells from human small and large intestine. A recent report by Wang et al. (2015) demonstrated homogeneous expansion of human fetal intestinal stem cells, providing a new culture system to understand self-renewing mechanisms of intestinal stem cells.

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Classical Th1 cells obtain colitogenicity by co-existence of RORγt-expressing T cells in experimental colitis

Saigusa Keiichiro, Hisamatsu Tadakazu, Handa Tango, Sujino Tomohisa, Mikami Yohei, Hayashi Atsushi, Mizuno Shinta, Takeshita Kozue, Sato Toshiro, Matsuoka Katsuyoshi, Kanai Takanori

Inflammatory Bowel Diseases 20 （ 10 ） 1820 - 1827 2014年10月

研究論文（学術雑誌）, 共著,  ISSN  1078-0998

　概要を見る

Background: Both Th1 and Th17 cell types are involved in the pathogenesis of chronic intestinal inflammation. We recently demonstrated that retinoid-related orphan receptor gamma t (RORγt)-expressing Th17 cells are progenitor cells for alternative Th1 cells, which have the potential to induce colitis. However, the involvement of classical Th1 (cTh1) cells generated directly from naive T cells without RORγt expression in the pathogenesis of colitis remains poorly understood. Methods: We performed a series of in vivo experiments using a murine chronic colitis model induced by adoptive transfer of splenic CD4+CD45RBhigh T cells obtained from wild-type, RORγt&lt;sup&gt;gfp/gfp&lt;/sup&gt;, or RORγt&lt;sup&gt;gfp/+&lt;/sup&gt; mice into RAG-2&lt;sup&gt;-/-&lt;/sup&gt; mice. Results: RAG-2&lt;sup&gt;-/-&lt;/sup&gt; mice receiving transfer of in vitro-manipulated RORγt&lt;sup&gt;gfp/gfp&lt;/sup&gt; Th1 cells developed colitis. RAG-2&lt;sup&gt;-/-&lt;/sup&gt; mice co-transferred with splenic CD4+CD45RB&lt;sup&gt;high&lt;/sup&gt; T cells obtained from wild-type mice and RORγt&lt;sup&gt;gfp/gfp&lt;/sup&gt; mice developed colitis with a significant increase in RORγt&lt;sup&gt;gfp/gfp&lt;/sup&gt; cTh1 cell numbers when compared with noncolitic mice transferred with splenic CD4+CD45RB&lt;sup&gt;high&lt;/sup&gt; T cells obtained from RORγt&lt;sup&gt;gfp/gfp&lt;/sup&gt; mice. Furthermore, RAG-2&lt;sup&gt;-/-&lt;/sup&gt; mice transferred with in vivo-manipulated RORγt&lt;sup&gt;gfp/gfp&lt;/sup&gt; cTh1 cells developed colitis with a significant increase in RORγt&lt;sup&gt;gfp/gfp&lt;/sup&gt; cTh1 cell numbers. Conclusions: These findings indicate that both alternative Th1 cells and cTh1 cells have the potential to be colitogenic in an adaptive transfer model. The development of cTh1 cells was dependent on the co-existence of RORγt-expressing T cells, suggesting a critical role for the interactions of these cell types in the development of chronic intestinal inflammation.

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KLF5 regulates the integrity and oncogenicity of intestinal stem cells

Nakaya Takeo, Ogawa Seishi, Manabe Ichiro, Tanaka Masami, Sanada Masashi, Sato Toshiro, Taketo Makoto M., Nakao Kazuki, Clevers Hans, Fukayama Masashi, Kuroda Masahiko, Nagai Ryozo

Cancer Research 74 （ 10 ） 2882 - 2891 2014年05月

研究論文（学術雑誌）, 共著,  ISSN  0008-5472

　概要を見る

The intestinal epithelium maintains homeostasis by a self-renewal process involving resident stem cells, including Lgr5+ crypt-base columnar cells, but core mechanisms and their contributions to intestinal cancer are not fully defined. In this study, we examined a hypothesized role for KLF5, a zinc-finger transcription factor that is critical to maintain the integrity of embryonic and induced pluripotent stem cells, in intestinal stem-cell integrity and cancer in the mouse. Klf5 was indispensable for the integrity and oncogenic transformation of intestinal stem cells. In mice, inducible deletion of Klf5 in Lgr5+ stem cells suppressed their proliferation and survival in a manner associated with nuclear localization of β-catenin (Catnb), generating abnormal apoptotic cells in intestinal crypts. Moreover, production of lethal adenomas and carcinomas by specific expression of an oncogenic mutant of β-catenin in Lgr5+ stem cells was suppressed completely by Klf5 deletion in the same cells. Given that activation of the Wnt/β-catenin pathway is the most frequently altered pathway in human colorectal cancer, our results argue that KLF5 acts as a fundamental core regulator of intestinal oncogenesis at the stem-cell level, and they suggest KLF5 targeting as a rational strategy to eradicate stem-like cells in colorectal cancer.

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Macrophages and dendritic cells emerge in the liver during intestinal inflammation and predispose the liver to inflammation

Mikami Yohei, Mizuno Shinta, Nakamoto Nobuhiro, Hayashi Atsushi, Sujino Tomohisa, Sato Toshiro, Kamada Nobuhiko, Matsuoka Katsuyoshi, Hisamatsu Tadakazu, Ebinuma Hirotoshi, Hibi Toshifumi, Yoshimura Akihiko, Kanai Takanori

PLoS One 9 （ 1 ）  2014年01月

共著

　概要を見る

The liver is a physiological site of immune tolerance, the breakdown of which induces immunity. Liver antigen-presenting cells may be involved in both immune tolerance and activation. Although inflammatory diseases of the liver are frequently associated with inflammatory bowel diseases, the underlying immunological mechanisms remain to be elucidated. Here we report two murine models of inflammatory bowel disease: RAG-2^-/- mice adoptively transferred with CD4⁺CD45RB^high T cells; and IL-10 ^-/- mice, accompanied by the infiltration of mononuclear cells in the liver. Notably, CD11b^-CD11c^lowPDCA-1⁺ plasmacytoid dendritic cells (DCs) abundantly residing in the liver of normal wild-type mice disappeared in colitic CD4⁺CD45RB^high T cell-transferred RAG-2^-/- mice and IL-10^-/- mice in parallel with the emergence of macrophages (Mφs) and conventional DCs (cDCs). Furthermore, liver Mφ/cDCs emerging during intestinal inflammation not only promote the proliferation of naïve CD4⁺ T cells, but also instruct them to differentiate into IFN-γ-producing Th1 cells in vitro. The emergence of pathological Mφ/cDCs in the liver also occurred in a model of acute dextran sulfate sodium (DSS)-induced colitis under specific pathogen-free conditions, but was canceled in germ-free conditions. Last, the Mφ/cDCs that emerged in acute DSS colitis significantly exacerbated Fas-mediated hepatitis. Collectively, intestinal inflammation skews the composition of antigen-presenting cells in the liver through signaling from commensal bacteria and predisposes the liver to inflammation.

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Continuous low-dose irradiation by I-125 seeds induces apoptosis of gastric cancer cells regardless of histological origin

Takabayashi Kaoru, Kashiwagi Kazuhiro, Kawata Tetsuya, Sato Toshiro, Matsuoka Katsuyoshi, Hisamatsu Tadakazu, Takaishi Hiromasa, Hibi Toshifumi, Ogata Haruhiko, Yahagi Naohisa, Kitagawa Yuko, Shigematsu Naoyuki, Kanai Takanori

Cancer Biology and Therapy 15 （ 1 ） 81 - 88 2014年01月

研究論文（学術雑誌）, 共著,  ISSN  1538-4047

　概要を見る

The efficacy of conventional radiation therapy for gastric cancer is controversial. In this study, we evaluated the in vitro and in vivo effects of continuous low-dose-rate irradiation by I-125 seeds on different histological types of gastric cancer cell lines. Three human gastric cancer cell lines (MKN74, MKN45, and NUGC4) were treated with or without continuous low-dose irradiation by I-125 seeds in vitro and in vivo. Cell viability, apoptosis, caspase-3 assay, and cell-cycle distribution were examined in vitro. Body weight and tumor volumes of BALB/c nude mice bearing MKN74, MKN45, and NUGC4 gastric cancer xenografts were measured, and in vivo cell proliferation and apoptosis assays were performed by Ki67 and TUNEL staining, respectively. Continuous low-dose-rate irradiation by I-125 seeds reduced cell viability and induced cell apoptosis through the activation of caspase-3, and led to the accumulation of cells in the G2/M phase in vitro. It also suppressed the growth of gastric cancer xenografts in nude mice, while inhibiting cell proliferation and inducing apoptosis as demonstrated by Ki67 and TUNEL staining. Therefore, our data suggest that continuous low-dose-rate irradiation by I-125 seeds could be a promising new option for gastric cancer treatment, regardless of histological origin.

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Transformation of intestinal stem cells into gastric stem cells on loss of transcription factor Cdx2

Simmini Salvatore, Bialecka Monika, Huch Meritxell, Kester Lennart, Van De Wetering Marc, Sato Toshiro, Beck Felix, Van Oudenaarden Alexander, Clevers Hans, Deschamps Jacqueline

Nature Communications 5 2014年

研究論文（学術雑誌）, 共著,  ISSN  2041-1723

　概要を見る

The endodermal lining of the adult gastro-intestinal tract harbours stem cells that are responsible for the day-to-day regeneration of the epithelium. Stem cells residing in the pyloric glands of the stomach and in the small intestinal crypts differ in their differentiation programme and in the gene repertoire that they express. Both types of stem cells have been shown to grow from single cells into 3D structures (organoids) in vitro. We show that single adult Lgr5-positive stem cells, isolated from small intestinal organoids, require Cdx2 to maintain their intestinal identity and are converted cell-autonomously into pyloric stem cells in the absence of this transcription factor. Clonal descendants of Cdx2 null small intestinal stem cells enter the gastric differentiation program instead of producing intestinal derivatives. We show that the intestinal genetic programme is critically dependent on the single transcription factor encoding gene Cdx2.

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Cross-talk between ROR_γt ⁺ innate lymphoid cells and intestinal macrophages induces mucosal IL-22 production In Crohn's disease

Mizuno Shinta, Mikami Yohei, Kamada Nobuhiko, Handa Tango, Hayashi Atsushi, Sato Toshiro, Matsuoka Katsuyoshi, Matano Mami, Ohta Yuki, Sugita Akira, Koganei Kazutaka, Sahara Rikisaburo, Takazoe Masakazu, Hisamatsu Tadakazu, Kanai Takanori

Inflammatory Bowel Diseases 20 （ 8 ） 1426 - 1434 2014年

研究論文（学術雑誌）, 共著,  ISSN  1078-0998

　概要を見る

Background: Interleukin (IL)-22-producing ROR_γt ⁺ innate lymphoid cells (ILCs) play a pivotal role in intestinal immunity. Recent reports demonstrated that ILCs contribute to mucosal protection and intestinal inflammation in mice. In humans, numbers of ROR _γt
⁺ ILCs are significantly increased in the intestine of patients with Crohn's disease (CD), suggesting that ILCs may be associated with intestinal inflammation in CD. However, the mechanism by which ILCs are regulated in the intestine of patients with CD is poorly understood. This study aimed to determine the activation mechanism of intestinal ILCs in patients with CD. Methods: CD45+ lineage marker-ILCs were isolated from intestinal lamina propria of patients with CD. ILCs were then subdivided into 4 distinct populations based on the expression of CD56 and CD127. Purified ILC subsets were cocultured with intestinal CD14+ macrophages, and IL-22 production was evaluated. Results: CD127⁺CD56^- and CD127 ⁺CD56⁺ ILC, but not CD127^-CD56⁺ or CD127^-CD56^- ILC, subsets expressed ROR_γt and produced IL-22. IL-22 production by these ILC subsets was enhanced when ILCs were cocultured with intestinal macrophages. IL-23 or cell-to-cell contact was required for macrophage-mediated activation of ILCs. IL-22 production by ILCs was perturbed in inflamed mucosa compared with noninflamed mucosa. IL-22 induced the expression of Reg1_α and Claudin-1 in human intestinal epithelial organoids. Conclusions: ROR_γt
⁺ ILCs might enhance mucosal barrier function through the upregulation of Reg1 _α through production of IL-22. Although CD14⁺ macrophages augment intestinal inflammation in patients with CD, macrophages also promote a negative feedback pathway through the activation of IL-22 production by ROR_γt
⁺ ILCs.

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Culturing intestinal stem cells

Fujii Masayuki, Sato Toshiro

Frontiers in Genetics 5 （ JUN ）  2014年

研究論文（学術雑誌）, 共著,  ISSN  1664-8021

　概要を見る

Recent advance of sequencing technology has revealed genetic alterations in colorectal cancer (CRC). The biological function of recurrently mutated genes has been intensively investigated through mouse genetic models and CRC cell lines. Although these experimental models may not fully reflect biological traits of human intestinal epithelium, they provided insights into the understanding of intestinal stem cell self-renewal, leading to the development of novel human intestinal organoid culture system. Intestinal organoid culture enabled to expand normal or tumor epithelial cells in vitro retaining their stem cell self-renewal and multiple differentiation. Gene manipulation of these cultured cells may provide an attractive tool for investigating genetic events involved in colorectal carcinogenesis.

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Mucosal healing with oral tacrolimus is associated with favorable medium- and long-term prognosis in steroid-refractory/dependent ulcerative colitis patients

Miyoshi Jun, Matsuoka Katsuyoshi, Inoue Nagamu, Hisamatsu Tadakazu, Ichikawa Riko, Yajima Tomoharu, Okamoto Susumu, Naganuma Makoto, Sato Toshiro, Kanai Takanori, Ogata Haruhiko, Iwao Yasushi, Hibi Toshifumi

Journal of Crohn's and Colitis 7 （ 12 ）  2013年12月

研究論文（学術雑誌）, 共著,  ISSN  1873-9946

　概要を見る

Background: Oral administration of tacrolimus is an effective remission induction therapy for steroid-refractory/dependent ulcerative colitis (UC). Aim: This study aimed to evaluate the short- as well as medium- and long-term effectiveness of tacrolimus therapy. Methods: The medical records of 51 patients treated with tacrolimus for UC at our hospital between July 2009 and December 2011 were reviewed retrospectively. Clinical remission and improvement were defined as a Lichtiger score of 4 or less and as a Lichtiger score of ≤. 10 and a reduction in the score of ≥. 3 compared with the baseline score, respectively. Endoscopic findings were evaluated based on the endoscopic activity index and Mayo endoscopic score. Results: The clinical effectiveness combining clinical remission and improvement was observed in 62.7% of the patients at 3. months. Thirty-six patients underwent colonoscopy at 3. months, and 12 (33.3%) and 10 patients (27.8%) showed Mayo endoscopic scores of 0 and 1, respectively. On Kaplan-Meier analysis, the overall percentage of event-free survivors, who did not require colectomy nor switching to other induction therapy such as infliximab, was 73.0% at 6. months, 49.9% at 1. year, and 37.8% at 2. years. Patients with a Mayo endoscopic score of 0-1 at 3. months showed significantly better medium- and long-term prognosis than those with a score of 2-3 (p&lt;. 0.01). All adverse events, including infections in 2 patients, were reversible. Conclusions: Tacrolimus therapy was effective for inducing clinical and endoscopic remission of steroid-refractory/dependent UC. Endoscopic improvement was associated with favorable medium- and long-term prognosis.

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Unlimited in vitro expansion of adult bi-potent pancreas progenitors through the Lgr5/R-spondin axis

Huch Meritxell, Bonfanti Paola, Boj Sylvia F., Sato Toshiro, Loomans Cindy J M, Van De Wetering Marc, Sojoodi Mozhdeh, Li Vivian S W, Schuijers Jurian, Gracanin Ana, Ringnalda Femke, Begthel Harry, Hamer Karien, Mulder Joyce, Van Es Johan H., De Koning Eelco, Vries Robert G J, Heimberg Harry, Clevers Hans

EMBO Journal 32 （ 20 ） 2708 - 2721 2013年10月

研究論文（学術雑誌）, 共著,  ISSN  0261-4189

　概要を見る

Lgr5 marks adult stem cells in multiple adult organs and is a receptor for the Wnt-agonistic R-spondins (RSPOs). Intestinal, stomach and liver Lgr5 + stem cells grow in 3D cultures to form ever-expanding organoids, which resemble the tissues of origin. Wnt signalling is inactive and Lgr5 is not expressed under physiological conditions in the adult pancreas. However, we now report that the Wnt pathway is robustly activated upon injury by partial duct ligation (PDL), concomitant with the appearance of Lgr5 expression in regenerating pancreatic ducts. In vitro, duct fragments from mouse pancreas initiate Lgr5 expression in RSPO1-based cultures, and develop into budding cyst-like structures (organoids) that expand five-fold weekly for &gt;40 weeks. Single isolated duct cells can also be cultured into pancreatic organoids, containing Lgr5 stem/progenitor cells that can be clonally expanded. Clonal pancreas organoids can be induced to differentiate into duct as well as endocrine cells upon transplantation, thus proving their bi-potentiality.

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[Intestinal stem cells].

佐藤　俊朗

生化学 （公益社団法人日本生化学会）  85 （ 9 ） 743 - 748 2013年09月

研究論文（学術雑誌）, 査読有り,  ISSN  0037-1017

Establishment of novel prediction system of intestinal absorption in humans using human intestinal tissues

Miyake Masateru, Toguchi Hajime, Nishibayashi Toru, Higaki Kazutaka, Sugita Akira, Koganei Kazutaka, Kamada Nobuhiko, Kitazume Mina T., Hisamatsu Tadakazu, Sato Toshiro, Okamoto Susumu, Kanai Takanori, Hibi Toshifumi

Journal of Pharmaceutical Sciences 102 （ 8 ） 2564 - 2571 2013年08月

研究論文（学術雑誌）, 共著,  ISSN  0022-3549

　概要を見る

The objective of this study was to establish a novel prediction system of drug absorption in humans by utilizing human intestinal tissues. Based on the transport index (TI), a newly defined parameter, calculated by taking account of the change in drug concentrations because of precipitation on the apical side and the amounts accumulated in the tissue and transported to the basal side, the absorbability of drugs in rank order as well as the fraction of dose absorbed (Fa) in humans were estimated. Human intestinal tissues taken from ulcerative colitis or Crohn's disease patients were mounted in a mini-Ussing chamber and transport studies were performed to evaluate the permeation of drugs, including FD-4, a very low permeable marker, atenolol, a low permeable marker, and metoprolol, a high permeable marker. Although apparent permeability coefficients calculated by the conventional equation did not reflect human Fa values for FD-4, atenolol, and metoprolol, TI values were well correlated with Fa values, which are described by 100 · [1 - e ^{- f · (TI - α)}]. Based on this equation, Fa values in humans for other test drugs were predicted successfully, indicating that our new system utilizing human intestinal tissues would be valuable for predicting oral drug absorption in humans.

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A single strain of Clostridium butyricum induces intestinal IL-10-producing macrophages to suppress acute experimental colitis in mice

Hayashi Atsushi, Sato Toshiro, Kamada Nobuhiko, Mikami Yohei, Matsuoka Katsuyoshi, Hisamatsu Tadakazu, Hibi Toshifumi, Roers Axel, Yagita Hideo, Ohteki Toshiaki, Yoshimura Akihiko, Kanai Takanori

Cell Host and Microbe 13 （ 6 ） 711 - 722 2013年06月

研究論文（学術雑誌）, 共著,  ISSN  1931-3128

　概要を見る

Imbalance in gut bacterial composition provokes host proinflammatory responses causing diseases such as colitis. Colonization with a mixture of Clostridium species from clusters IV and XIVa was shown to suppress colitis through the induction of IL-10-producing regulatory T (Treg) cells. We demonstrate that a distinct Clostridium strain from cluster I, Clostridium butyricum (CB), prevents acute experimental colitis in mice through induction of IL-10, an anti-inflammatory cytokine. However, while CB treatment had no effect on IL-10 production by T cells, IL-10-producing F4/80⁺CD11b ⁺CD11c^int macrophages accumulated in the inflamed mucosa after CB treatment. CB directly triggered IL-10 production by intestinal macrophages in inflamed mucosa via the TLR2/MyD88 pathway. The colitis-preventing effect of CB was negated in macrophage-specific IL-10-deficient mice, suggesting that induction of IL-10 by intestinal macrophages is crucial for the probiotic action of CB. Collectively, CB promotes IL-10 production by intestinal macrophages in inflamed mucosa, thereby preventing experimental colitis in mice.

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TGR5 signalling inhibits the production of pro-inflammatory cytokines by in vitro differentiated inflammatory and intestinal macrophages in Crohn's disease

Yoneno Kazuaki, Hisamatsu Tadakazu, Shimamura Katsuyoshi, Kamada Nobuhiko, Ichikawa Riko, Kitazume Mina T., Mori Maiko, Uo Michihide, Namikawa Yuka, Matsuoka Katsuyoshi, Sato Toshiro, Koganei Kazutaka, Sugita Akira, Kanai Takanori, Hibi Toshifumi

Immunology 139 （ 1 ） 19 - 29 2013年05月

研究論文（学術雑誌）, 共著,  ISSN  0019-2805

　概要を見る

Bile acids (BAs) play important roles not only in lipid metabolism, but also in signal transduction. TGR5, a transmembrane receptor of BAs, is an immunomodulative factor, but its detailed mechanism remains unclear. Here, we aimed to delineate how BAs operate in immunological responses via the TGR5 pathway in human mononuclear cell lineages. We examined TGR5 expression in human peripheral blood monocytes, several types of in vitro differentiated macrophages (Mφ{symbol}s) and dendritic cells. Mφ{symbol}s differentiated with macrophage colony-stimulating factor and interferon-γ (Mγ-Mφ{symbol}s), which are similar to the human intestinal lamina propria CD14⁺ Mφ{symbol}s that contribute to Crohn's disease (CD) pathogenesis by production of pro-inflammatory cytokines, highly expressed TGR5 compared with any other type of differentiated Mφ{symbol} and dendritic cells. We also showed that a TGR5 agonist and two types of BAs, deoxycholic acid and lithocholic acid, could inhibit tumour necrosis factor-α production in Mγ-Mφ{symbol}s stimulated by commensal bacterial antigen or lipopolysaccharide. This inhibitory effect was mediated by the TGR5-cAMP pathway to induce phosphorylation of c-Fos that regulated nuclear factor-κB p65 activation. Next, we analysed TGR5 levels in lamina propria mononuclear cells (LPMCs) obtained from the intestinal mucosa of patients with CD. Compared with non-inflammatory bowel disease, inflamed CD LPMCs contained more TGR5 transcripts. Among LPMCs, isolated CD14⁺ intestinal Mφ{symbol}s from patients with CD expressed TGR5. In isolated intestinal CD14⁺ Mφ{symbol}s, a TGR5 agonist could inhibit tumour necrosis factor-α production. These results indicate that TGR5 signalling may have the potential to modulate immune responses in inflammatory bowel disease.

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IL-22-Producing RORγt-Dependent Innate Lymphoid Cells Play a Novel Protective Role in Murine Acute Hepatitis

Matsumoto Atsuhiro, Kanai Takanori, Mikami Yohei, Chu Po Sung, Nakamoto Nobuhiro, Ebinuma Hirotoshi, Saito Hidetsugu, Sato Toshiro, Yagita Hideo, Hibi Toshifumi

PLoS One 8 （ 4 ）  2013年04月

研究論文（学術雑誌）, 共著, 査読有り

　概要を見る

<p>Retinoid-related orphan receptor (ROR) γt is known to be related to the development and function of various immunological compartments in the liver, such as Th17 cells, natural killer T (NKT) cells, and innate lymphoid cells (ILCs). We evaluated the roles of RORγt-expressing cells in mouse acute hepatitis model using RORγt deficient (RORγt^-/-) mice and RAG-2 and RORγt double deficient (RAG-2^-/- × RORγt^-/-) mice. Acute hepatitis was induced in mice by injection with carbon tetrachloride (CCl₄), to investigate the regulation of liver inflammation by RORγt-expressing cells. We detected RORC expression in three compartments, CD4⁺ T cells, NKT cells, and lineage marker-negative SCA-1⁺Thy1^high ILCs, of the liver of wild type (WT) mice. CCl₄-treated RORγt^-/- mice developed liver damage in spite of lack of RORγt-dependent cells, but with reduced infiltration of macrophages compared with WT mice. In this regard, ILCs were significantly decreased in RAG-2^-/- × RORγt^-/- mice that lacked T and NKT cells. Surprisingly, RAG-2^-/- × RORγt^-/- mice developed significantly severer CCl₄-induced hepatitis compared with RAG-2^-/- mice, in accordance with the fact that hepatic ILCs failed to produce IL-22. Lastly, anti-Thy1 monoclonal antibody (mAb), but not anti-NK1.1 mAb or anti-asialo GM1 Ab administration exacerbated liver damage in RAG-2^-/- mice with the depletion of liver ILCs. Collectively, hepatic RORγt-dependent ILCs play a part of protective roles in hepatic immune response in mice.</p>

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In vitro expansion of single Lgr5 + liver stem cells induced by Wnt-driven regeneration

Huch Meritxell, Dorrell Craig, Boj Sylvia F., Van Es Johan H., Li Vivian S W, Van De Wetering Marc, Sato Toshiro, Hamer Karien, Sasaki Nobuo, Finegold Milton J., Haft Annelise, Vries Robert G., Grompe Markus, Clevers Hans

Nature 494 （ 7436 ） 247 - 250 2013年02月

研究論文（学術雑誌）, 共著,  ISSN  0028-0836

　概要を見る

The Wnt target gene Lgr5 (leucine-rich-repeat-containing G-protein-coupled receptor 5) marks actively dividing stem cells in Wnt-driven, self-renewing tissues such as small intestine and colon, stomach and hair follicles. A three-dimensional culture system allows long-term clonal expansion of single Lgr5⁺ stem cells into transplantable organoids (budding cysts) that retain many characteristics of the original epithelial architecture. A crucial component of the culture medium is the Wnt agonist RSPO1, the recently discovered ligand of LGR5. Here we show that Lgr5-lacZ is not expressed in healthy adult liver, however, small Lgr5-LacZ + cells appear near bile ducts upon damage, coinciding with robust activation of Wnt signalling. As shown by mouse lineage tracing using a new Lgr5-IRES-creERT2 knock-in allele, damage-induced Lgr5⁺ cells generate hepatocytes and bile ducts in vivo. Single Lgr5⁺ cells from damaged mouse liver can be clonally expanded as organoids in Rspo1-based culture medium over several months. Such clonal organoids can be induced to differentiate in vitro and to generate functional hepatocytes upon transplantation into Fah-/-mice. These findings indicate that previous observations concerning Lgr5⁺ stem cells in actively self-renewing tissues can also be extended to damage-induced stem cells in a tissue with a low rate of spontaneous proliferation.

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Dendritic cells administered intrarectally penetrate the intestinal barrier to break intestinal tolerance via Th2-medeiated colitis in mice

Handa Tango, Kanai Takanori, Sato Toshiro, Mikami Yohei, Sujino Tomohisa, Hayashi Atsushi, Mizuno Shinta, Matsumoto Atsuhiro, Hisamatsu Tadakazu, Hibi Toshifumi

Immunology Letters 150 （ 1-2 ） 123 - 129 2013年02月

研究論文（学術雑誌）, 共著,  ISSN  0165-2478

　概要を見る

Intestinal lamina propria dendritic cells (LPDCs) in mice are known to extend dendrites between the intestinal epithelia and the luminal side when processing luminal antigens. We conducted intrarectal cell transfer experiments of bone marrow-derived dendritic cells (BMDCs) in mice to assess dendritic cell penetration of the intestine. Intrarectally administered GFP⁺ BMDCs localized in the colonic LP within 3h and the spleen within 12h after administration. 72h after administration, recipient C57BL/6 mice showed acute diarrhea, and administration of BMDCs (once weekly for 3 weeks) induced intestinal inflammation with increased numbers of recipient macrophages and CD4⁺ T cells exhibiting a Th2-mediated immune response. These results demonstrate that DCs actively communicate across the intestinal barrier, and highlight a potential technique for controlling colonic immune tolerance.

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Primary mouse small intestinal epithelial cell cultures

Sato Toshiro, Clevers Hans

Methods in Molecular Biology （Springer）  945   319 - 328 2013年

共著, 査読有り

　概要を見る

<p>The intestinal epithelium is the most rapidly self-renewing tissue in adult mammals. We have recently shown that Lgr5 (Leucine-rich repeat-containing G protein-coupled receptor) is expressed in intestinal stem cells by an in vivo genetic lineage tracing strategy. In the past, extensive efforts have been made to establish primary small intestinal culture systems. However, no defined, reproducible and robust culture system had been developed. To establish such a system, we screened for optimal growth factor combinations based on genetic evidence of self-renewal regulation, differentiation, and carcinogenesis of intestinal stem cells. Here, we describe methods that we have established for the isolation and culture of primary small intestinal epithelial stem cells. In this culture system, isolated crypts form organoid structures with a histological hierarchy recapitulating in vivo small intestinal epithelium. Single isolated Lgr5+ intestinal stem cells also form these organoid structures, in which stem cells are maintained by self-renewal and give rise to all lineages of the intestinal epithelium. This culture system is particularly useful for studying the regulation of intestinal stem cell self-renewal and differentiation.</p>

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Growing self-organizing mini-guts from a single intestinal stem cell

Sato Toshiro, Clevers Hans

Science 340 （ 6137 ） 1190 - 1194 2013年

研究論文（学術雑誌）, 共著,  ISSN  0036-8075

　概要を見る

Recent examples have highlighted how stem cells have the capability to initiate morphogenesis in vitro; that is, to generate complex structures in culture that closely parallel their in vivo counterparts. Lgr5, the receptor for the Wnt-agonistic R-spondins, marks stem cells in multiple adult organs of mice and humans. In R-spondin-based three-dimensional cultures, these Lgr5 stem cells can grow into ever-expanding epithelial organoids that retain their original organ identity. Single Lgr5 stem cells derived from the intestine can be cultured to build epithelial structures that retain hallmarks of the in vivo epithelium. Here, we review the mechanisms that support this notable example of self-organization and discuss applications of this technology for stem cell research, disease modeling (e.g., for colorectal cancer and cystic fibrosis), and regenerative medicine.

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Dysregulated balance of retinoid-related orphan receptor γt-dependent innate lymphoid cells is involved in the pathogenesis of chronic DSS-induced colitis

Kimura Kayoko, Kanai Takanori, Hayashi Atsushi, Mikami Yohei, Sujino Tomohisa, Mizuno Shinta, Handa Tango, Matsuoka Katsuyoshi, Hisamatsu Tadakazu, Sato Toshiro, Hibi Toshifumi

Biochemical and Biophysical Research Communications 427 （ 4 ） 694 - 700 2012年11月

研究論文（学術雑誌）, 共著,  ISSN  0006-291X

　概要を見る

Retinoid-related orphan receptor (ROR) γt-expressing and IL-22-producing NKp46⁺ innate lymphoid (ILC22) cells reside in the lamina propria of the intestine in mice, suggesting that ILC22 cells contribute to host defense during intestinal damage in models of colitis in mice. Nevertheless, another set of pathological interferon (IFN)-γ and/or IL-17A-producing innate lymphoid cells (ILC1 and ICL17) may participate in the pathogenesis in different models of colitis. We here showed that RORγt⁺IL-22⁺ ILC22 cells were localized in Thy-1^highSCA-1^high and/or Thy-1^highSCA-1^low subpopulations of the intestine in normal and dextran sodium sulfate (DSS)-induced colitic RORγt-sufficient Rag-2^-/- mice. RORγt-deficient Rag-2^-/- mice developed more severe DSS-induced colitis accompanied with lower expression of REG3β and REG3γ in the colon, but with a lower ratio and absolute number of IFN-γ-producing ILC1 cells as compared to control RORγt-sufficient Rag-2^-/- mice. Collectively, not only the presence of ILC22 cells but also the balance of protective and pathogenic ILCs may be involved in the prevention of colitis.

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Dll1 + secretory progenitor cells revert to stem cells upon crypt damage

Van Es Johan H., Sato Toshiro, Van De Wetering Marc, Lyubimova Anna, Yee Nee Annie Ng, Gregorieff Alex, Sasaki Nobuo, Zeinstra Laura, Van Den Born Maaike, Korving Jeroen, Martens Anton C M, Barker Nick, Van Oudenaarden Alexander, Clevers Hans

Nature Cell Biology 14 （ 10 ） 1099 - 1104 2012年10月

研究論文（学術雑誌）, 共著,  ISSN  1465-7392

　概要を見る

Lgr5 + intestinal stem cells generate enterocytes and secretory cells. Secretory lineage commitment requires Notch silencing. The Notch ligand Dll1 is expressed by a subset of immediate stem cell daughters. Lineage tracing in Dll1 GFP-ires-CreERT2 knock-in mice reveals that single Dll1 high cells generate small, short-lived clones containing all four secretory cell types. Lineage specification thus occurs in immediate stem cell daughters through Notch lateral inhibition. Cultured Dll1 high cells form long-lived organoids (mini-guts) on brief Wnt3A exposure. When Dll1 high cells are genetically marked before tissue damage, stem cell tracing events occur. Thus, secretory progenitors exhibit plasticity by regaining stemness on damage.

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On the biomechanics of stem cell niche formation in the gut - Modelling growing organoids

Buske Peter, Przybilla Jens, Loeffler Markus, Sachs Norman, Sato Toshiro, Clevers Hans, Galle Joerg

FEBS Journal 279 （ 18 ） 3475 - 3487 2012年09月

研究論文（学術雑誌）, 共著,  ISSN  1742-464X

　概要を見る

In vitro culture of intestinal tissue has been attempted for decades. Only recently did Sato et al. [Sato, T., Vries, R. G., Snippert, H. J., van de Wetering, M., Barker, N., Stange, D. E., van Es, J. H., Abo, A., Kujala, P., Peters, P. J., et al. (2009) Nature459, 262-265] succeed in establishing long-term intestinal culture, demonstrating that cells expressing the Lgr5 gene can give rise to organoids with crypt-like domains similar to those found in vivo. In these cultures, Paneth cells provide essential signals supporting stem cell function. We have recently developed an individual cell-based computational model of the intestinal tissue [Buske, P., Galle, J., Barker, N., Aust, G., Clevers, H. &amp; Loeffler, M. (2011) PLoS Comput Biol7, e1001045]. The model is capable of quantitatively reproducing a comprehensive set of experimental data on intestinal cell organization. Here, we present a significant extension of this model that allows simulation of intestinal organoid formation in silico. For this purpose, we introduce a flexible basal membrane that assigns a bending modulus to the organoid surface. This membrane may be re-organized by cells attached to it depending on their differentiation status. Accordingly, the morphology of the epithelium is self-organized. We hypothesize that local tissue curvature is a key regulatory factor in stem cell organization in the intestinal tissue by controlling Paneth cell specification. In simulation studies, our model closely resembles the spatio-temporal organization of intestinal organoids. According to our results, proliferation-induced shape fluctuations are sufficient to induce crypt-like domains, and spontaneous tissue curvature induced by Paneth cells can control cell number ratios. Thus, stem cell expansion in an organoid depends sensitively on its biomechanics. We suggest a number of experiments that will enable new insights into mechano-transduction in the intestine, and suggest model extensions in the field of gland formation.

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CCR9⁺ macrophages are required for eradication of peritoneal bacterial infections and prevention of polymicrobial sepsis

Mizukami Takahiro, Kanai Takanori, Mikami Yohei, Hayashi Atsushi, Doi Tomomitsu, Handa Tango, Matsumoto Atsuhiro, Jun Li, Matsuoka Katsuyoshi, Sato Toshiro, Hisamatsu Tadakazu, Hibi Toshifumi

Immunology Letters 147 （ 1-2 ） 75 - 79 2012年09月

研究論文（学術雑誌）, 共著,  ISSN  0165-2478

　概要を見る

Sepsis is a systemic inflammatory response to infection associated with multiple organ dysfunction syndrome and a high mortality rate. In septic shock induced by severe peritonitis, early response of peritoneal macrophages against infected microbes is vital in preventing the spread of infection. We found that the mucosal homing receptor CCR9, is induced in peritoneal macrophages in response to inflammatory stimulation. We used a cecal ligation and puncture (CLP) model of sepsis to determine the role of CCR9 with respect to peritoneal macrophages, and controlling peritoneal infection and systemic inflammation. CCR9^-/- mice showed aggravated septic shock with higher mortality rates compared with wild-type (WT) mice. Six hours after CLP, CCR9^-/- mice demonstrated a greater inflammatory response. This was associated with higher production of inflammatory cytokines, such as IL-6, TNF and IP-10 in peritoneal lavage compared with WT mice. Although the numbers of peritoneal bacteria were elevated in CCR9^-/- mice subjected to CLP compared with WT mice, this was normalized in CCR9^-/- mice subjected to CLP through the adoptive transfer of WT peritoneal macrophages. We conclude that CCR9 is required for recruitment of peritoneal macrophages in the steady state to control systemic sepsis during early phases of peritoneal infection.

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The use of infliximab in the prevention of postsurgical recurrence in polysurgery Crohn's disease patients

Sakuraba Atsushi, Sato Toshiro, Matsukawa Hidehiko, Okamoto Susumu, Takaishi Hiromasa, Ogata Haruhiko, Iwao Yasushi, Hibi Toshifumi

International Journal of Colorectal Disease 27 （ 7 ） 947 - 952 2012年07月

研究論文（学術雑誌）, 共著,  ISSN  0179-1958

　概要を見る

Purpose Crohn's disease (CD) commonly recurs after surgery, and a number of patients need repeated surgery, especially smokers and those with repeated surgeries or penetrating disease. Whether infliximab prevents postsurgical recurrence in high-risk CD remains unknown. In the present pilot openlabeled study, we investigated the safety and efficacy of scheduled infliximab, which was started early after surgery, in maintaining remission of CD patients who have undergonemultiple surgeries due to penetrating disease. Methods Eleven patients (nine male, two female; age range, 26-48 years) who had undergone repeated surgeries (median, 4; range, 2-5) for penetrating disease were enrolled. Two to 4 weeks after surgery, the patients were started on intravenous infliximab (5 mg/kg) at an 8-week interval. The primary end points were the proportion of patients in clinical remission at the end of the study, the rate of endoscopic/ radiologic remission at 24 months, and the rate of adverse effects. Results One patient dropped out due to non-compliance, and ten patients were eligible for analysis. Clinical remission was maintained in six of ten patients (60.0%) at the end of the study. At 24 months, four out of ten patients were in endoscopic or radiological remission (40.0%). Two patients experienced adverse effects (18.2%), one of whom elected to withdraw from the study. Conclusion The findings of no major safety concern and possible clinical benefit in our study suggest that further investigation of infliximab as a treatment for prevention of postsurgical recurrence in high-risk CD is warranted.

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Intermittent Granulocyte and Monocyte Apheresis Versus Mercaptopurine for Maintaining Remission of Ulcerative Colitis

Sakuraba Atsushi, Sato Toshiro, Morohoshi Yuichi, Matsuoka Katsuyoshi, Okamoto Susumu, Inoue Nagamu, Takaishi Hiromasa, Ogata Haruhiko, Iwao Yasushi, Hibi Toshifumi

Therapeutic Apheresis and Dialysis 16 （ 3 ） 213 - 218 2012年06月

研究論文（学術雑誌）, 共著,  ISSN  1744-9979

　概要を見る

The effect of granulocyte and monocyte adsorption apheresis (GMA) on prevention of relapse of ulcerative colitis (UC) is not clear. This was a pilot open-labeled, prospective, randomized, unblinded study to compare the tolerability and efficacy of intermittent GMA (once every 2weeks) with mercaptopurine to maintain remission of UC. Twenty-one patients with UC, who had achieved remission by induction therapies were randomly assigned to receive either intermittent GMA (N=10) or oral mercaptopurine (0.5mg/kg per day; N=11). The study period was 24months. The rate of the patients maintaining remission and the incidences of adverse effects were compared between the two groups. At 24months, seven of 10 patients (70.0%) on intermittent GMA and seven of 11 patients (63.6%, P=1.00) on oral mercaptopurine were still in remission. Three patients relapsed in each group. One patient taking mercaptopurine, but none receiving intermittent GMA, dropped out because of adverse effects. Intermittent therapy with GMA was well tolerated and a substantial proportion of patients maintained remission. Intermittent GMA therapy in maintaining remission of UC merits further investigation.

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Functional engraftment of colon epithelium expanded in vitro from a single adult Lgr5 + stem cell

Yui Shiro, Nakamura Tetsuya, Sato Toshiro, Nemoto Yasuhiro, Mizutani Tomohiro, Zheng Xiu, Ichinose Shizuko, Nagaishi Takashi, Okamoto Ryuichi, Tsuchiya Kiichiro, Clevers Hans, Watanabe Mamoru

Nature Medicine 18 （ 4 ） 618 - 623 2012年04月

研究論文（学術雑誌）, 共著,  ISSN  1078-8956

　概要を見る

Adult stem-cell therapy holds promise for the treatment of gastrointestinal diseases. Here we describe methods for long-term expansion of colonic stem cells positive for leucine-rich repeat containing G protein-coupled receptor 5 (Lgr5 ⁺ cells) in culture. To test the transplantability of these cells, we reintroduced cultured GFP ⁺ colon organoids into superficially damaged mouse colon. The transplanted donor cells readily integrated into the mouse colon, covering the area that lacked epithelium as a result of the introduced damage in recipient mice. At 4 weeks after transplantation, the donor-derived cells constituted a single-layered epithelium, which formed self-renewing crypts that were functionally and histologically normal. Moreover, we observed long-term (&gt;6 months) engraftment with transplantation of organoids derived from a single Lgr5 ⁺ colon stem cell after extensive in vitro expansion. These data show the feasibility of colon stem-cell therapy based on the in vitro expansion of a single adult colonic stem cell.

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Cdx2 determines the fate of postnatal intestinal endoderm

Stringer Emma J., Duluc Isabelle, Saandi Thoueiba, Davidson Irwin, Bialecka Monika, Sato Toshiro, Barker Nick, Clevers Hans, Pritchard Catrin A., Winton Doug J., Wright Nicholas A., Freund Jean Noel, Deschamps Jacqueline, Beck Felix

Development 139 （ 3 ） 465 - 474 2012年02月

研究論文（学術雑誌）, 共著,  ISSN  0950-1991

　概要を見る

Knock out of intestinal Cdx2 produces different effects depending upon the developmental stage at which this occurs. Early in development it produces histologically ordered stomach mucosa in the midgut. Conditional inactivation of Cdx2 in adult intestinal epithelium, as well as specifically in the Lgr5-positive stem cells, of adult mice allows long-term survival of the animals but fails to produce this phenotype. Instead, the endodermal cells exhibit cell-autonomous expression of gastric genes in an intestinal setting that is not accompanied by mesodermal expression of Barx1, which is necessary for gastric morphogenesis. Cdx2- negative endodermal cells also fail to express Sox2, a marker of gastric morphogenesis. Maturation of the stem cell niche thus appears to be associated with loss of ability to express positional information cues that are required for normal stomach development. Cdx2-negative intestinal crypts produce subsurface cystic vesicles, whereas untargeted crypts hypertrophy to later replace the surface epithelium. These observations are supported by studies involving inactivation of Cdx2 in intestinal crypts cultured in vitro. This abolishes their ability to form long-term growing intestinal organoids that differentiate into intestinal phenotypes. We conclude that expression of Cdx2 is essential for differentiation of gut stem cells into any of the intestinal cell types, but they maintain a degree of cell-autonomous plasticity that allows them to switch on a variety of gastric genes.

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Controlled gene expression in primary Lgr5 organoid cultures

Koo Bon Kyoung, Stange Daniel E., Sato Toshiro, Karthaus Wouter, Farin Henner F., Huch Meritxell, Van Es Johan H., Clevers Hans

Nature Methods 9 （ 1 ） 81 - 83 2012年01月

研究論文（学術雑誌）, 共著,  ISSN  1548-7091

　概要を見る

The study of gene function in endodermal epithelia such as of stomach, small intestine and colon relies heavily on transgenic approaches. Establishing such animal models is laborious, expensive and time-consuming. We present here a method based on Cre recombinaseinducible retrovirus vectors that allows the conditional manipulation of gene expression in primary mouse organoid culture systems.

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Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett's epithelium

Sato Toshiro, Stange Daniel E., Ferrante Marc, Vries Robert G J, Van Es Johan H., Van Den Brink Stieneke, Van Houdt Winan J., Pronk Apollo, Van Gorp Joost, Siersema Peter D., Clevers Hans

Gastroenterology 141 （ 5 ） 1762 - 1772 2011年11月

研究論文（学術雑誌）, 共著,  ISSN  0016-5085

　概要を見る

Background &amp; Aims: We previously established long-term culture conditions under which single crypts or stem cells derived from mouse small intestine expand over long periods. The expanding crypts undergo multiple crypt fission events, simultaneously generating villus-like epithelial domains that contain all differentiated types of cells. We have adapted the culture conditions to grow similar epithelial organoids from mouse colon and human small intestine and colon. Methods: Based on the mouse small intestinal culture system, we optimized the mouse and human colon culture systems. Results: Addition of Wnt3A to the combination of growth factors applied to mouse colon crypts allowed them to expand indefinitely. Addition of nicotinamide, along with a small molecule inhibitor of Alk and an inhibitor of p38, were required for long-term culture of human small intestine and colon tissues. The culture system also allowed growth of mouse Apc-deficient adenomas, human colorectal cancer cells, and human metaplastic epithelia from regions of Barrett's esophagus. Conclusions: We developed a technology that can be used to study infected, inflammatory, or neoplastic tissues from the human gastrointestinal tract. These tools might have applications in regenerative biology through ex vivo expansion of the intestinal epithelia. Studies of these cultures indicate that there is no inherent restriction in the replicative potential of adult stem cells (or a Hayflick limit) ex vivo.

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Isolation and in vitro expansion of human colonic stem cells

Jung Peter, Sato Toshiro, Merlos-Suárez Anna, Barriga Francisco M., Iglesias Mar, Rossell David, Auer Herbert, Gallardo Mercedes, Blasco Maria A., Sancho Elena, Clevers Hans, Batlle Eduard

Nature Medicine 17 （ 10 ） 1225 - 1227 2011年10月

研究論文（学術雑誌）, 共著,  ISSN  1078-8956

　概要を見る

Here we describe the isolation of stem cells of the human colonic epithelium. Differential cell surface abundance of ephrin type-B receptor 2 (EPHB2) allows the purification of different cell types from human colon mucosa biopsies. The highest EPHB2 surface levels correspond to epithelial colonic cells with the longest telomeres and elevated expression of intestinal stem cell (ISC) marker genes. Moreover, using culturing conditions that recreate the ISC niche, a substantial proportion of EPHB2-high cells can be expanded in vitro as an undifferentiated and multipotent population.

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Paneth cells constitute the niche for Lgr5 stem cells in intestinal crypts

Sato Toshiro, Van Es Johan H., Snippert Hugo J., Stange Daniel E., Vries Robert G., Van Den Born Maaike, Barker Nick, Shroyer Noah F., Van De Wetering Marc, Clevers Hans

Nature 469 （ 7330 ） 415 - 418 2011年01月

研究論文（学術雑誌）,  ISSN  0028-0836

　概要を見る

Homeostasis of self-renewing small intestinal crypts results from neutral competition between Lgr5 stem cells, which are small cycling cells located at crypt bottoms. Lgr5 stem cells are interspersed between terminally differentiated Paneth cells that are known to produce bactericidal products such as lysozyme and cryptdins/defensins. Single Lgr5-expressing stem cells can be cultured to form long-lived, self-organizing crypt-villus organoids in the absence of non-epithelial niche cells. Here we find a close physical association of Lgr5 stem cells with Paneth cells in mice, both in vivo and in vitro. CD24
⁺ Paneth cells express EGF, TGF-γ ±, Wnt3 and the Notch ligand Dll4, all essential signals for stem-cell maintenance in culture. Co-culturing of sorted stem cells with Paneth cells markedly improves organoid formation. This Paneth cell requirement can be substituted by a pulse of exogenous Wnt. Genetic removal of Paneth cells in vivo results in the concomitant loss of Lgr5 stem cells. In colon crypts, CD24
⁺ cells residing between Lgr5 stem cells may represent the Paneth cell equivalents. We conclude that Lgr5 stem cells compete for essential niche signals provided by a specialized daughter cell, the Paneth cell.

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Lgr5+ve Stem Cells Drive Self-Renewal in the Stomach and Build Long-Lived Gastric Units In Vitro

Barker Nick, Huch Meritxell, Kujala Pekka, van de Wetering Marc, Snippert Hugo J., van Es Johan H., Sato Toshiro, Stange Daniel E., Begthel Harry, van den Born Maaike, Danenberg Esther, van den Brink Stieneke, Korving Jeroen, Abo Arie, Peters Peter J., Wright Nick, Poulsom Richard, Clevers Hans

Cell Stem Cell 6 （ 1 ） 25 - 36 2010年01月

研究論文（学術雑誌）, 共著,  ISSN  1934-5909

　概要を見る

The study of gastric epithelial homeostasis and cancer has been hampered by the lack of stem cell markers and in vitro culture methods. The Wnt target gene Lgr5 marks stem cells in the small intestine, colon, and hair follicle. Here, we investigated Lgr5 expression in the stomach and assessed the stem cell potential of the Lgr5^+ve cells by using in vivo lineage tracing. In neonatal stomach, Lgr5 was expressed at the base of prospective corpus and pyloric glands, whereas expression in the adult was predominantly restricted to the base of mature pyloric glands. Lineage tracing revealed these Lgr5^+ve cells to be self-renewing, multipotent stem cells responsible for the long-term renewal of the gastric epithelium. With an in vitro culture system, single Lgr5^+ve cells efficiently generated long-lived organoids resembling mature pyloric epithelium. The Lgr5 stem cell marker and culture method described here will be invaluable tools for accelerating research into gastric epithelial renewal, inflammation/infection, and cancer.

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Intestinal crypt homeostasis results from neutral competition between symmetrically dividing Lgr5 stem cells

Snippert Hugo J., van der Flier Laurens G., Sato Toshiro, van Es Johan H., van den Born Maaike, Kroon-Veenboer Carla, Barker Nick, Klein Allon M., van Rheenen Jacco, Simons Benjamin D., Clevers Hans

Cell 143 （ 1 ） 134 - 144 2010年

研究論文（学術雑誌）, 共著,  ISSN  0092-8674

　概要を見る

Intestinal stem cells, characterized by high Lgr5 expression, reside between Paneth cells at the small intestinal crypt base and divide every day. We have carried out fate mapping of individual stem cells by generating a multicolor Cre-reporter. As a population, Lgr5^hi stem cells persist life-long, yet crypts drift toward clonality within a period of 1-6 months. We have collected short- and long-term clonal tracing data of individual Lgr5^hi cells. These reveal that most Lgr5^hi cell divisions occur symmetrically and do not support a model in which two daughter cells resulting from an Lgr5^hi cell division adopt divergent fates (i.e., one Lgr5^hi cell and one transit-amplifying [TA] cell per division). The cellular dynamics are consistent with a model in which the resident stem cells double their numbers each day and stochastically adopt stem or TA fates. Quantitative analysis shows that stem cell turnover follows a pattern of neutral drift dynamics.

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Th1/Th17 Immune Response Is Induced by Mesenteric Lymph Node Dendritic Cells in Crohn's Disease

Sakuraba Atsushi, Sato Toshiro, Kamada Nobuhiko, Kitazume Mina, Sugita Akira, Hibi Toshifumi

Gastroenterology 137 （ 5 ） 1736 - 1745 2009年11月

研究論文（学術雑誌）, 共著,  ISSN  0016-5085

　概要を見る

Background &amp; Aims: Dendritic cells (DCs) possess the most potent ability to induce acquired immunity. However, their involvement in the pathogenesis of Crohn's disease (CD) has not yet been determined. We aimed to establish the immune status of mesenteric lymph nodes, the major gut-associated lymphoid tissue, and isolated DCs and determine their involvement in the pathogenesis of CD. Methods: CD4 ⁺ T cells and DCs were isolated from mesenteric lymph nodes of CD, ulcerative colitis, and normal control. The immune status of CD4 ⁺ T cells was analyzed by cytokine production and transcriptional profile. Surface phenotype of DCs was analyzed by flow cytometry. Cytokine production by myeloid DCs was analyzed by real-time polymerase chain reaction and exogenous bacterial stimulation. Immune stimulating activity of DCs was determined by mixed lymphocyte reaction. Results: In CD, mesenteric lymph node CD4 ⁺ T cells produced higher amounts of interferon-γ and interleukin (IL)-17 compared with ulcerative colitis and normal control, and this was dictated by increased T-bet and retinoic acid-related orphan receptor-γ expression. Three subtypes of DCs, myeloid DC, plasmacytoid DC, and mature DC, were identified in all groups. When stimulated with exogenous bacterial derivative, myeloid DCs from CD produced a higher amount of IL-23 and a lower amount of IL-10. Myeloid DCs from CD induced stronger T helper cell (Th)1 immune response in mixed lymphocyte reaction compared with those from ulcerative colitis and normal control. Conclusions: Our findings revealed that mesenteric lymph node is the key pathogenic location of CD elicited by the unique cytokine milieu produced by DCs leading to a dysregulated Th1/Th17 immune response.

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Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche

Sato Toshiro, Vries Robert G., Snippert Hugo J., Van De Wetering Marc, Barker Nick, Stange Daniel E., Van Es Johan H., Abo Arie, Kujala Pekka, Peters Peter J., Clevers Hans

Nature 459 （ 7244 ） 262 - 265 2009年05月

研究論文（学術雑誌）, 共著,  ISSN  0028-0836

　概要を見る

The intestinal epithelium is the most rapidly self-renewing tissue in adult mammals. We have recently demonstrated the presence of about six cycling Lgr5 + stem cells at the bottoms of small-intestinal crypts. Here we describe the establishment of long-term culture conditions under which single crypts undergo multiple crypt fission events, while simultanously generating villus-like epithelial domains in which all differentiated cell types are present. Single sorted Lgr5 + stem cells can also initiate these crypt-villus organoids. Tracing experiments indicate that the Lgr5 + stem-cell hierarchy is maintained in organoids. We conclude that intestinal crypt-villus units are self-organizing structures, which can be built from a single stem cell in the absence of a non-epithelial cellular niche.

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Granulocyte and monocyte adsorption apheresis therapy modulates monocyte-derived dendritic cell function in patients with ulcerative colitis

Iwakami Yuko, Sakuraba Atsushi, Sato Toshiro, Takada Yasuhiro, Izumiya Motoko, Ichikawa Hitoshi, Hibi Toshifumi

Therapeutic Apheresis and Dialysis 13 （ 2 ） 138 - 146 2009年04月

研究論文（学術雑誌）, 共著,  ISSN  1744-9979

　概要を見る

The aim of this study was to elucidate the molecular mechanisms responsible for the therapeutic effects of granulocyte and monocyte adsorption apheresis (GMA). We investigated the alterations in circulating monocyte subsets and monocyte-derived dendritic cell (moDC) function after GMA therapy in ulcerative colitis (UC) patients. Eighteen patients with UC were enrolled: 14 patients were responders, and 4 patients were non-responders. Peripheral venous blood was obtained within 5 min before and 5 min after GMA therapy. Flow cytometric analysis for monocyte markers (CD14/CD16) was then performed. Monocyte-derived dendritic cells were obtained and alterations in their phenotype were analyzed by flow cytometry. Their function was also analyzed in a mixed lymphocyte reaction assay between allo-naïve T lymphocytes. Flow cytometric analysis for intracellular interferon (IFN)-γ (T-helper 1 cells) and interleukin (IL)-4 (T-helper 2 cells) was then performed for the stimulated T lymphocytes. In patients who responded to GMA, the average numbers of monocytes, especially CD16⁺ monocytes, were significantly decreased after therapy (P &lt; 0.05). In responders, post-GMA moDCs expressed significantly lower CD80 and B7-DC, which are one of the stimulation and maturation markers of dendritic cells, compared to pre-GMA moDCs. CD83, CD86 and human leukocyte antigen-DR also showed a tendency to decrease. In responders, naïve T lymphocytes stimulated with post-GMA moDCs produced significantly less IFN-γ and IL-4 compared to those stimulated with pre-GMA moDCs. The results of our study show that some of the immunosuppressive effects of GMA therapy may be associated with the modulation of monocyte subsets and moDC function.

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Unique CD14⁺ intestinal macrophages contribute to the pathogenesis of Crohn disease via IL-23/IFN-γ axis

Kamada Nobuhiko, Hisamatsu Tadakazu, Okamoto Susumu, Chinen Hiroshi, Kobayashi Taku, Sato Toshiro, Sakuraba Atsushi, Kitazume Mina T., Sugita Akira, Koganei Kazutaka, Akagawa Kiyoko S., Hibi Toshifumi

Journal of Clinical Investigation 118 （ 6 ） 2269 - 2280 2008年06月

研究論文（学術雑誌）, 共著,  ISSN  0021-9738

　概要を見る

Intestinal macrophages play a central role in regulation of immune responses against commensal bacteria. In general, intestinal macrophages lack the expression of innate-immune receptor CD14 and do not produce proinflammatory cytokines against commensal bacteria. In this study, we identified what we believe to be a unique macrophage subset in human intestine. This subset expressed both macrophage (CD14, CD33, CD68) and DC markers (CD205, CD209) and produced larger amounts of proinflammatory cytokines, such as IL-23, TNF-α, and IL-6, than typical intestinal resident macrophages (CD14 ^-CD33⁺ macrophages). In patients with Crohn disease (CD), the number of these CD14+ macrophages were significantly increased compared with normal control subjects. In addition to increased numbers of cells, these cells also produced larger amounts of IL-23 and TNF-α compared with those in normal controls or patients with ulcerative colitis. In addition, the CD14 ⁺ macrophages contributed to IFN-γ production rather than IL-17 production by lamina propria mononuclear cells (LPMCs) dependent on IL-23 and TNF-α. Furthermore, the IFN-γ produced by LPMCs triggered further abnormal macrophage differentiation with an IL-23-hyperproducing phenotype. Collectively, these data suggest that this IL-23/IFN-γ-positive feedback loop induced by abnormal intestinal macrophages contributes to the pathogenesis of chronic intestinal inflammation in patients with CD.

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Current View

Scoville David H., Sato Toshiro, He Xi C., Li Linheng

Gastroenterology 134 （ 3 ） 849 - 864 2008年03月

研究論文（学術雑誌）, 共著,  ISSN  0016-5085

　概要を見る

Studies using mice have yielded significant amounts of information regarding signaling pathways, such as Wnt, bone morphogenic protein, PtdIns(3,4,5) kinase, and Notch, involved in intestinal development and homeostasis, including stem cell regulation and lineage specification and maturation. However, attempts to model signals definitively that control intestinal stem cells have been difficult because of a long-standing and recently reenergized debate surrounding their location. Although crypt-based columnar cells have been recently shown to display self-renewal and multipotential capacity, a large body of evidence supports long-term label-retaining cells, located on average at the +4 position just above the Paneth cells, as putative stem cells. Herein, we propose that both these cell types represent true intestinal stem cells maintained in different states (quiescent vs actively cycling), presumably via interactions with different microenvironments. Finally, we review current findings regarding the roles of Wnt, bone morphogenic protein, PtdIns(3,4,5) kinase, and Notch pathways within the intestine.

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A pilot open-labeled prospective randomized study between weekly and intensive treatment of granulocyte and monocyte adsorption apheresis for active ulcerative colitis

Sakuraba Atsushi, Sato Toshiro, Naganuma Makoto, Morohoshi Yuichi, Matsuoka Katsuyoshi, Inoue Nagamu, Takaishi Hiromasa, Ogata Haruhiko, Iwao Yasushi, Hibi Toshifumi

Journal of Gastroenterology 43 （ 1 ） 51 - 56 2008年01月

研究論文（学術雑誌）, 共著,  ISSN  0944-1174

　概要を見る

Background. Recently, granulocyte and monocyte adsorption apheresis (GMA) has been shown to be effective for active ulcerative colitis (UC). Its original weekly treatment schedule is effective in about 70% of active UC. However, it takes about 3-4 weeks to achieve remission, and the efficacy of a more frequent treatment schedule has not been elucidated yet. We performed a pilot open-labeled prospective, randomized, controlled study comparing weekly and an intensive treatment schedule with three treatment sessions per week in the first 2 weeks. Methods. Thirty active UC patients with moderate disease activity were prospectively and randomly assigned to receive the original or the intensive treatment schedule for a total of ten sessions. The proportion of the patients achieving remission and the time to achieve remission among them was compared between the two groups. The incidences of adverse effects were also compared between the two groups. Results. The rate of inducing remission in the original and intensive treatment group was 66.7% and 80%, respectively (P = 0.25, NS). The time to achieve remission was 27.2 days in the original group and 10.7 days in the intensive group (P = 0.04). Adverse effects were observed in two patients in each groups (NS). Conclusions. Intensive treatment with GMAis an efficacious and safe treatment for active UC. Because it induces rapid remission, it may be a more ideal treatment regimen than the conventional weekly treatment.

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Lamina Propria c-kit⁺ Immune Precursors Reside in Human Adult Intestine and Differentiate Into Natural Killer Cells

Chinen Hiroshi, Matsuoka Katsuyoshi, Sato Toshiro, Kamada Nobuhiko, Okamoto Susumu, Hisamatsu Tadakazu, Kobayashi Taku, Hasegawa Hirotoshi, Sugita Akira, Kinjo Fukunori, Fujita Jiro, Hibi Toshifumi

Gastroenterology 133 （ 2 ） 559 - 573 2007年08月

研究論文（学術雑誌）, 共著,  ISSN  0016-5085

　概要を見る

Background &amp; Aims: Recent studies have revealed that murine intestinal mucosa contains several kinds of lineage markers (lin)^- c-kit⁺ immune precursor cells. However, immune precursors in the human adult intestine have not been studied extensively. Methods: Lamina propria mononuclear cells and intraepithelial lymphocytes from surgically resected human adult intestine were examined for the surface antigen expression and cytokine profile by immunohistochemistry and flow cytometry. The transcriptional profile of these cells was analyzed by reverse-transcription polymerase chain reaction. The phenotypic and functional characterization of the in vitro differentiating cells from the precursors was examined by flow cytometry. Results: We identified lin^- c-kit⁺ cells scattered throughout lamina propria of the human adult intestine. These intestinal immune precursors expressed CD34, CD38, CD33, interleukin-2Rα, and interleukin-7Rα, and they had much more abundant expression of Id2, PU.1, SpiB1, and lymphotoxin than thymocytes. The lin^- c-kit⁺ immune precursors mainly differentiated into CD56⁺ c-kit^dim cells during in vitro culture. These in vitro differentiating cells corresponded to intestinal natural killer (NK) cells, which had distinct characteristics from their peripheral counterparts, such as CD83 and integrin α_E expression, less cytotoxic activity, and higher interferon-γ production. Furthermore, both c-kit^dim cells and NK cells were increased in lamina propria of Crohn's disease, although there was no change for peripheral blood NK cells. Conclusions: The human intestine may have the unique NK cell differentiation system, which may contribute to maintenance of immune homeostasis in the intestine.

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PTEN-deficient intestinal stem cells initiate intestinal polyposis

He Xi C., Yin Tong, Grindley Justin C., Tian Qiang, Sato Toshiro, Tao W. Andy, Dirisina Raminarao, Porter-Westpfahl Kimberly S., Hembree Mark, Johnson Teri, Wiedemann Leanne M., Barrett Terrence A., Hood Leroy, Wu Hong, Li Linheng

Nature Genetics 39 （ 2 ） 189 - 198 2007年02月

研究論文（学術雑誌）, 共著,  ISSN  1061-4036

　概要を見る

Intestinal polyposis, a precancerous neoplasia, results primarily from an abnormal increase in the number of crypts, which contain intestinal stem cells (ISCs). In mice, widespread deletion of the tumor suppressor Phosphatase and tensin homolog (PTEN) generates hamartomatous intestinal polyps with epithelial and stromal involvement. Using this model, we have established the relationship between stem cells and polyp and tumor formation. PTEN helps govern the proliferation rate and number of ISCs and loss of PTEN results in an excess of ISCs. In PTEN-deficient mice, excess ISCs initiate de novo crypt formation and crypt fission, recapitulating crypt production in fetal and neonatal intestine. The PTEN-Akt pathway probably governs stem cell activation by helping control nuclear localization of the Wnt pathway effector β-catenin. Akt phosphorylates β-catenin at Ser552, resulting in a nuclear-localized form in ISCs. Our observations show that intestinal polyposis is initiated by PTEN-deficient ISCs that undergo excessive proliferation driven by Akt activation and nuclear localization of β-catenin.

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Cytomegalovirus is frequently reactivated and disappears without antiviral agents in ulcerative colitis patients

Matsuoka Katsuyoshi, Iwao Yasushi, Mori Takeshi, Sakuraba Atsushi, Yajima Tomoharu, Hisamatsu Tadakazu, Okamoto Susumu, Morohoshi Yuichi, Izumiya Motoko, Ichikawa Hitoshi, Sato Toshiro, Inoue Nagamu, Ogata Haruhiko, Hibi Toshifumi

American Journal of Gastroenterology 102 （ 2 ） 331 - 337 2007年02月

研究論文（学術雑誌）, 共著,  ISSN  0002-9270

　概要を見る

OBJECTIVE: The clinical significance of cytomegalovirus (CMV) reactivation complicating ulcerative colitis (UC) patients has been uncertain. It has therefore remained undetermined whether or not CMV reactivation should be treated in UC patients under immunosuppression. The aim of the study was to clarify the natural history of CMV reactivation in UC patients. METHODS: Sixty-nine UC patients with moderate to severe activity were enrolled in the study. All of the patients were treated with prednisolone, and/or immunosuppressants such as cyclosporine A. We sequentially monitored CMV reactivation every 2 wk up until 8 wk using the CMV antigenemia (Ag) assay and plasma quantitative real-time polymerase chain reaction (PCR) assay for CMV. RESULTS: Immunoglobulin (Ig) G for CMV was positive in 48 patients (69.6%) and negative in 21 patients (30.4%). CMV was reactivated in 25 patients out of the 48 seropositive patients (52.1%) during the study period. The CMV Ag and PCR values were low and none of the patients showed any evidence of CMV infection on biopsy specimens by hematoxylin and eosin staining. While gancylovir (GCV) was not used except in two patients, clinical outcomes including rates of remission and colectomy were not significantly different among the CMV reactivation-positive, -negative, and CMV IgG negative groups. Furthermore, CMV disappeared without GCV in most of the CMV reactivation-positive patients. CONCLUSIONS: CMV is frequently reactivated in active UC patients; however, it disappears without antiviral agents. Therefore, antiviral therapies should not be necessary for most UC patients with only CMV reactivation as long as CMV Ag values are low.

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Inhibition of neutrophil elastase prevents the development of murine dextran sulfate sodium-induced colitis

Morohoshi Yuichi, Matsuoka Katsuyoshi, Chinen Hiroshi, Kamada Nobuhiko, Sato Toshiro, Hisamatsu Tadakazu, Okamoto Susumu, Inoue Nagamu, Takaishi Hiromasa, Ogata Haruhiko, Iwao Yasushi, Hibi Toshifumi

Journal of Gastroenterology 41 （ 4 ） 318 - 324 2006年04月

研究論文（学術雑誌）, 共著,  ISSN  0944-1174

　概要を見る

Background: Neutrophil elastase (NE) is a major secretory product from activated neutrophils and a major contributor to tissue destruction. However, little is known about the pathogenic contribution of NE to ulcerative colitis (UC). This study was designed to investigate the contribution of NE by measuring NE activity in plasma and colonic mucosal tissue from UC patients and a murine acute colitis model, and to elucidate the therapeutic effect of the NE-specific inhibitor ONO-5046. Methods: The NE enzyme activities in plasma and colonic mucosal tissue from UC patients were directly measured using an enzyme-substrate reaction. Acute colitis was induced in mice by administration of 1.5% dextran sulfate sodium (DSS) for 5 days. DSS-induced colitis mice were then treated with ONO-5046 (50 mg/kg body weight) intraperitoneally twice a day. Results: In UC patients, the NE enzyme activity was significantly elevated in both the plasma and colonic mucosal tissue compared with healthy controls. In DSS-induced colitis mice, the NE enzyme activity increased in parallel with the disease development. ONO-5046 showed therapeutic effects in DSS-treated mice by significantly reducing weight loss and histological score. ONO-5046 suppressed the NE enzyme activities in both plasma and culture supernatant of colonic mucosa from DSS-induced colitis mice. Conclusions: ONO-5046, a specific NE inhibitor, prevented the development of DSS-induced colitis in mice. NE therefore represents a promising target for the treatment of UC patients.

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Abnormally differentiated subsets of intestinal macrophage play a key role in Th1-dominant chronic colitis through excess production of IL-12 and IL-23 in response to bacteria

Kamada Nobuhiko, Hisamatsu Tadakazu, Okamoto Susumu, Sato Toshiro, Matsuoka Katsuyoshi, Arai Kumiko, Nakai Takaaki, Hasegawa Akira, Inoue Nagamu, Watanabe Noriaki, Akagawa Kiyoko S., Hibi Toshifumi

Journal of Immunology 175 （ 10 ） 6900 - 6908 2005年11月

研究論文（学術雑誌）, 共著,  ISSN  0022-1767

　概要を見る

Disorders in enteric bacteria recognition by intestinal macrophages (Mφ) are strongly correlated with the pathogenesis of chronic colitis; however the precise mechanisms remain unclear. The aim of the current study was to elucidate the roles of Mφ in intestinal inflammation by using an IL-10-deficient (IL-10^-/-) mouse colitis model. GM-CSF-induced bone marrow-derived Mφ (GM-Mφ) and M-CSF-induced bone marrow-derived Mφ (M-Mφ) were generated from bone marrow CD11b⁺ cells. M-Mφ from IL-10^-/- mice produced abnormally large amounts of IL-12 and IL-23 upon stimulation with heat-killed whole bacteria Ags, whereas M-Mφ from wild-type (WT) mice produced large amounts of IL-10 but not IL-12 or IL-23. In contrast, IL-12 production by GM-Mφ was not significantly different between WT and IL-10^-/- mice. In ex vivo experiments, cytokine production ability of colonic lamina propria Mφ (CLPMφ) but not splenic Mφ from WT mice was similar to that of M-Mφ, and CLPMφ but not splenic Mφ from IL-10^-/- mice also showed abnormal IL-12p70 hyperproduction upon stimulation with bacteria. Surprisingly, the abnormal IL-12p70 hyperproduction from IL-10^-/- from IL-10^-/- mice was improved by IL-10 supplementation during the differentiation process. These results suggest that CLPMφ and M-Mφ act as anti-inflammatory Mφ and suppress excess inflammation induced by bacteria in WT mice. In IL-10 ^-/- mice, however, such Mφ subsets differentiated into an abnormal phenotype under an IL-10-deficient environment, and bacteria recognition by abnormally differentiated subsets of intestinal Mφ may lead to Th1-dominant colitis via IL-12 and IL-23 hyperproduction. Our data provide new insights into the intestinal Mφ to gut flora relationship in the development of colitis in IL-10^-/- mice.

Osteopontin/Eta-1 upregulated in Crohn's disease regulates the Th1 immune response

Sato T., Nakai T., Tamura N., Okamoto S., Matsuoka K., Sakuraba A., Fukushima T., Uede T., Hibi T.

Gut 54 （ 9 ） 1254 - 1262 2005年09月

ISSN  0017-5749

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Background and aims: The pathogenesis of Crohn's disease (CD), a chronic inflammatory bowel disease characterised by a Th1 immune response, remains unclear. Osteopontin (OPN) is a phosphoprotein known as an adhesive bone matrix protein. Recent studies have shown that OPN plays an important role in lymphocyte migration, granuloma formation, and interleukin 12 (IL-12) production. The present study investigated expression and the pathophysiological role of OPN in CD. Methods: Plasma OPN concentration was measured by enzyme linked immunosorbent assay. Expression of OPN in human intestinal mucosa was determined using reverse transcription-polymerase chain reaction and western blot, and localisation of OPN was examined by immunohistochemistry. Expression of integrin s, an OPN receptor, on lamina propria mononuclear cells (LPMC) was assessed by flow cytometry. Functional activation of OPN in LPMC was investigated by measuring the production of cytokines. Results: Plasma OPN concentration was significantly higher in patients with CD compared with normal controls or patients with ulcerative colitis (UC). OPN was upregulated in intestinal mucosa from UC and CD patients. OPN producing cells were epithelial or IgG producing plasma cells, or partial macrophages. OPN was detected in areas surrounding granuloma from mucosa in CD. Integrin βs expressing macrophages infiltrated inflamed mucosa in UC and CD; in contrast, there was no expression of integrin βs on intestinal macrophages in normal mucosa. OPN induced production of IL-12 from LPMC in CD but not in normal controls or UC. Conclusions: Increased OPN expression facilitates cytokine production and is closely involved in the Th1 immune response associated with CD.

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Nonpathogenic Escherichia coli strain Nissle 1917 prevents murine acute and chronic colitis

Kamada Nobuhiko, Inoue Nagamu, Hisamatsu Tadakazu, Okamoto Susumu, Matsuoka Katsuyoshi, Sato Toshiro, Chinen Hiroshi, Hong Kyong Su, Yamada Takaya, Suzuki Yumiko, Suzuki Tatsuo, Watanabe Noriaki, Tsuchimoto Kanji, Hibi Toshifumi

Inflammatory Bowel Diseases 11 （ 5 ） 455 - 463 2005年05月

ISSN  1078-0998

　概要を見る

Background: Nonpathogenic Escherichia coli strain Nissle1917 has been used as a probiotics in human inflammatory bowel disease; however, there are few reports examining its therapeutic effect on animal colitis models, and its therapeutic mechanisms remain unknown. The aim of this study was to elucidate the therapeutic effect and mechanism of Nissle1917 using murine acute and chronic colitis models. Methods: Two models were used. (1) Acute model: colitis was induced by administration of 1.3% dextran sodium sulfate for 7 days. Nissle1917 or phosphate-buffered saline were orally administered for 10 days. Mice were killed at day 10, and the colonic lesions were assessed macro- and microscopically. (2) Chronic model: IL-10^-/- mice were treated with Nissle1917 or phosphate-buffered saline for 8 weeks. After 8 weeks of treatment, mice were killed to assess the colonic lesions macro- and microscopically. In the acute dextran sodium sulfate colitis model, viable, heat-killed, or genomic DNA of Nissle1917 was orally administered for 10 days, and the therapeutic effect was assessed. Results: In the acute model, Nissle1917 ameliorated body weight loss, disease activity index, and macro- and microscopic damage. In the chronic model, it also suppressed the mucosal inflammatory findings and histologic damages. Moreover, heat-killed Nissle1917 or its genomic DNA alone also ameliorated the acute DSS colitis and viable bacteria macro- and microscopically. Conclusions: Nonpathogenic E. coli strain Nissle1917 prevents both acute and chronic colitis, and its anti-inflammatory effect is exhibited not only by viable bacteria but also by heat-killed bacteria or its DNA.

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T-bet upregulation and subsequent interleukin 12 stimulation are essential for induction or Th1 mediated immunopathology in Crohn's disease

Matsuoka K., Inoue N., Sato T., Okamoto S., Hisamatsu T., Kishi Y., Sakuraba A., Hitotsumatsu O., Ogata H., Koganei K., Fukushima T., Kanai T., Watanabe M., Ishii H., Hibi T.

Gut 53 （ 9 ） 1303 - 1308 2004年09月

ISSN  0017-5749

　概要を見る

Background and aims: Many lines of evidence suggest that T helper cell type 1 (Th1) immune responses predominate in Crohn's disease (CD). Recently, a novel transcription factor T-box expressed in T cells (T-bet) has been reported as the master regulator of Th1 development. This study was designed to investigate the role of T-bet and proinflammatory cytokines in Th1 mediated immunopathology in CD. Materials: CD4+ lamina propria mononuclear cells (LPMCs) were isolated from surgically resected specimens (CD, n = 10; ulcerative colitis (UC), n = 10; normal controls (NL), n = 5). Methods: (1) T-bet expression of CD4+ LPMCs was examined by quantitative real time polymerase chain reaction and western blotting. (2) T-bet expression of LPMCs stimulated by interleukin (IL)-12/IL-18 was analysed by western blotting. (3) Interferon γ (IFN-γ) production and T-bet expression of CD4+ peripheral blood mononuclear cells (PBMCs) were examined with or without stimulation by anti-CD3/CD28 monoclonal antibodies and/or IL-12. Results: (1) T-bet expression of CD4+ LPMCs was increased in CD compared with UC and NL. (2) Synergistically, augmentation of IFN-γ production by IL-12/IL-18 was independent of T-bet expression in LPMCs. (3) T-bet was induced by T cell receptor stimulation in CD4+ PBMCs. T-bet induction correlated with IFN-γ production and with augmentation of surface expressed IL-12 receptor β2. Conclusions: T-bet induction by antigenic stimulation and subsequent stimulation by macrophage derived IL-12/IL-18 are important for establishing Th1 mediated immunopathology in CD.

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Human intestinal epithelial cell-derived interleukin (IL)-18, along with IL2, IL-7 and IL-15, is a potent synergistic factor for the proliferation of intraepithelial lymphocytes

Okazawa A., Kanai T., Nakamaru K., Sato T., Inoue N., Ogata H., Iwao Y., Ikeda M., Kawamura T., Makita S., Uraushihara K., Okamoto R., Yamazaki M., Kurimoto M., Ishii H., Watanabe M., Hibi T.

Clinical and Experimental Immunology 136 （ 2 ） 269 - 276 2004年05月

ISSN  0009-9104

　概要を見る

Intestinal epithelial cell (IEC)-derived cytokines, such as stem cell factor (SCF), interleukin (IL)-7 and IL-15 are known to be required for the development of intestinal intraepithelial lymphocytes (IELs). A newly described cytokine, IL-18, has also been shown to be produced by intestinal epithelial cells. To demonstrate the functional effects of IL-18 on human IELs, we assessed IL-18/IL-18 receptor expression in IEC/IEL and proliferation following stimulation of intestinal IELs by IL-18. IL-18 transcripts were detected both in freshly isolated human colonie epithelial cells and in various colonie epithelial cell lines. IL-18 protein was also detected by ELISA and flow cytometric analysis using antihuman IL-18-specific monoclonal antibody (MoAb). Furthermore, IELs constitutively expressed the IL-18 receptor in addition to the IL-2 and IL-7 receptors. More importantly, IL-18 augmented significant proliferative responses of IEL in combination with IL-2, IL-7 and IL-15 both in the presence and in absence of anti-CD3 MoAb. These results suggest that IL-18 might play a crucial role in the proliferation and maintenance of intestinal IELs.

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Hyperexpression of Inducible Costimulator and Its Contribution on Lamina Propria T Cells in Inflammatory Bowel Disease

Sato Toshiro, Kanai Takanori, Watanabe Mamoru, Sakuraba Atsushi, Okamoto Susumu, Nakai Takaaki, Okazawa Akira, Inoue Nagamu, Totsuka Teruji, Yamazaki Motomi, Kroczek Richard A., Fukushima Tsuneo, Ishii Hiromasa, Hibi Toshifumi

Gastroenterology 126 （ 3 ） 829 - 839 2004年03月

ISSN  0016-5085

　概要を見る

Background &amp; Aims: To investigate the role of inducible costimulator (ICOS), a new member of the CD28 family involved in regulation of T-cell activation and chronic intestinal inflammation, we assessed its expression and functional role in patients with inflammatory bowel disease (IBD). Methods: Expression of ICOS, CD28, and cytotoxic T-lymphocyte antigen (CTLA) 4 on intestinal lamina propria mononuclear cells (LPMC) from patients with ulcerative colitis (UC), Crohn's disease (CD), and normal controls was determined using flow cytometry and immunohistochemistry. Expressions of the ICOS ligand, B7h, on lamina propria B cells, macrophages, and epithelial cells (EC) in the intestinal mucosa were also determined using flow cytometry. The functional costimulatory effect of ICOS on LPMC was assessed by the proliferative response and cytokine production. Results: CD4⁺ LPMC expressing ICOS was significantly increased in the inflamed mucosa of IBD patients but not in inflammatory or normal controls. B7h was also significantly up-regulated on B cells, macrophages, and EC in inflamed mucosa of IBD patients. Proliferative responses of anti-CD3/ICOS costimulation were significantly higher compared with those of anti-CD3 monoclonal antibody (mAb) alone. Anti-CD3/ICOS-stimulated-LPMC from UC secreted significantly increased amounts of interleukin (IL)-5 among the 3 groups. In contrast, anti-CD3/ICOS-stimulated-LPMC from CD secreted significantly increased amounts of interferon (IFN)-γ in the presence of IL-12. Conclusions: Highly expressed ICOS in activated CD4⁺ LPMC of IBD patients contributes to the dysregulated immune responses in IBD. Because ICOS hyperexpression was limited to inflammatory sites in IBD patients, ICOS would be a feasible therapeutic target for the treatment of IBD.

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Contrasting action of IL-12 and IL-18 in the development of dextran sodium sulphate colitis in mice

Takagi H., Kanai T., Okazawa A., Kishi Y., Sato T., Takaishi H., Inoue N., Ogata H., Iwao Y., Hoshino K., Takeda K., Akira S., Watanabe M., Ishii H., Hibi Toshifumi

Scandinavian Journal of Gastroenterology 38 （ 8 ） 837 - 844 2003年08月

ISSN  0036-5521

　概要を見る

Background: Interleukin (IL)-12 and IL-18 are major interferon (IFN)-γ-inducing factors that collaborate with each other. The present study was conducted to determine the distinct roles of IL-12 and IL-18 in the development of dextran sulphate sodium (DSS) colitis in mice. Methods: Colitis was induced in IL-12p35^-/-, IL-18^-/-, IL-18 receptor ^-/- and control mice with DSS. Clinical and histopathological analysis was conducted using survival rate, weight loss score, diarrhoea score, bloody stool score and histological score. In addition, cytokine production by lamina propria mononuclear cells (LPMCs) was examined using the specific enzyme-linked immunoassay. Results: IL-12p35^-/- mice developed only a mild disease associated with no lethality and few histopathological abnormalities. In contrast, IL-18^-/- and IL-18R^-/- mice developed more severe colitis associated with high lethality and more histopathological abnormalities compared with control mice. LPMCs from DSS-fed IL-18^-/- mice produced significantly higher amounts of IFN-γ, while LPMCs from DSS-fed IL-12^-/- mice produced lower amounts of IFN-γ and tumour necrosis factor (TNF)-α compared with control mice. Conclusion: These results suggest that IL-18 might function with manners different from IL-12 at some pathological conditions in the development of colitis.

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下部消化管疾患

佐藤俊朗，日比紀文

内科 90 （ 6 ） 1024-1027 2002年12月

研究論文（学術雑誌）, 共著

炎症性腸疾患

佐藤俊朗，日比紀文

Annual review 消化器 2002   239-243 2002年

研究論文（学術雑誌）, 共著

The citrus flavonoid, nobiletin, inhibits peritoneal dissemination of human gastric carcinoma in SCID mice

Minagawa Akiko, Otani Yoshihide, Kubota Teturo, Wada Norihito, Furukawa Toshiharu, Kumai Koichirou, Kameyama, Kaori, Okada Yasunori, Fujii Masato, Yano Masamichi, Sato Takashi, Ito Akira, Kitajima Masaki

Japanese Journal of Cancer Research 92 （ 12 ） 1322-1328 2001年12月

研究論文（学術雑誌）, 共著

HLA class-I-restricted and colon-specific cytotoxic T cells from lamina propria lymphocytes of patients with ulcerative colitis

Sunagawa Takesh, Yonamine Yoshimasa, Kinjo Fukunori, Watanabe Mamoru, Hibi Toshifumi, Saito Atsushi

Journal of Clinical Immunology 21 （ 6 ） 381-389 2001年11月

研究論文（学術雑誌）, 共著

Macrophage-derived IL-18-mediated intestinal inflammation in the murine model of Crohn's disease

Kanai Takanori, Watanabe Mamoru, Okazawa Akira, Sato Toshiro, Yamazaki Motomi, Okamoto Susumu, Ishii Hiromasa, Totsuka Teruji, Iiyama Ryoichi, Okamoto Ryuichi, Ikeda Masao, Kurimoto Masashi, Takeda Kiyoshi, Akira Shizuo, Hibi Toshifumi

Gastroenterology 121 （ 4 ） 875 - 888 2001年10月

研究論文（学術雑誌）, 共著,  ISSN  0016-5085

　概要を見る

<p>Background &amp; Aims: Crohn's disease (CD) is associated with an increased number of infiltrating macrophages, which release a variety of proinflammatory cytokines. Interleukin (IL)-18 has been implicated in the modulation of mucosal CD4 ⁺ T cells towards Th1 responses, which are implicated in the pathogenesis of CD. Here we assess the role of macrophages and of IL-18 in the murine model of intestinal inflammation that mimics the immunologic characteristics of human CD. Methods: Colitis was induced in C57BL/6 mice immunized with 2,4,6-trinitrobenzene sulfonic acid (TNBS) followed by rectal administration of TNBS in ethanol. Mice were treated with either an antibody directed against macrophages conjugated to the ribosome-inactivating protein saporin (anti-Mac-1-saporin) or with a neutralizing antibody against IL-18. In addition, we assessed whether an identical TNBS immunization/challenge protocol could induce colitis in IL-18 ^-/- mice. Results: The colonic mucosa of TNBS-treated mice was marked by infiltration of Mac-1-positive macrophages and up-regulation of IL-18. The administration of the anti-Mac-1-saporin antibody or the neutralizing anti-IL-18 antibody resulted in a dramatic attenuation of mucosal inflammation in this model. In addition, TNBS was unable to induce significant colitis in the IL-18 ^-/- mice. Conclusions: Our data underscore the pivotal role of macrophages, and the macrophage-derived IL-18, in the establishment of TNBS-induced colitis in mice. Our results highlight the potential use of therapy directed against IL-18 in the treatment of patients with CD.</p>

Restricted VH Gene usage in lamina propria B Cells producing anticolon antibody from patients with ulcerative colitis

Inoue Nagamu, Watanabe Mamoru, Sato Toshirou, Okazawa Akira, Yamazaki Motomi, Kanai Takanori, Ogata Haruhiko, Iwao Yasushi, Ishii Hiromasa，Hibi Toshifumi

Gastroenterology　 121 （ 1 ） 15-23 2001年07月

研究論文（学術雑誌）, 共著

内視鏡的に摘出しえた回腸アニサキス症の1例

佐藤俊朗，今枝博之，緒方晴彦，岩男泰，細田泰雄，長沼誠，江崎俊彦，中澤敦，井上詠，日比紀文，石井裕正，向井万起男

Progress of Digestive Endoscopy 58   112-113 2001年06月

研究論文（学術雑誌）, 共著

Interleukin-18 and Crohn's disease

Kanai Takanori, Watanabe Mamoru, Okazawa Akira, Sato Toshiro, Hibi Toshifumi

Digestion 63 （ suppl. 1 ） 37 - 42 2001年01月

研究論文（学術雑誌）, 共著,  ISSN  0012-2823

　概要を見る

The etiology and pathogenesis of inflammatory bowel disease remains an area under intense investigation. Crohn's disease (CD) is characterized by a marked accumulation of activated Th1 type CD4+ T cells and macrophages in inflamed intestinal mucosa. IL-18 is a recently described cytokine that mainly exists in activated macrophages and shares biological activities with IL-12 in driving the development of Th1 type CD4+ T cells by inducing interferon-γ. To clarify the role of IL-18 in intestinal inflammation in CD, we assessed the functional role of IL-18 in regulating intestinal mucosal lymphocytes in human CD and murine CD model.</p>

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Restricted V_H gene usage in lamina propria B cells producing anticolon antibody from patients with ulcerative colitis

Inoue Nagamu, Watanabe Mamoru, Sato Toshiro, Okazawa Akira, Yamazaki Motomi, Kanai Takanori, Ogata Haruhiko, Iwao Yasushi, Ishii Hiromasa, Hibi Toshifumi

Gastroenterology 121 （ 1 ） 15 - 23 2001年

ISSN  0016-5085

　概要を見る

Background &amp; Aims: Autoimmune responses against colonic epithelium may play a role in the development of colonic inflammation associated with ulcerative colitis (UC). In this study, we established and characterized B-cell lines and clones that produced anticolon antibody from inflamed colonic mucosa of UC subjects. Methods: B-cell lines were generated through Epstein-Barr virus transformation of lamina propria lymphocytes (LPLs) from colonic mucosa and peripheral blood lymphocytes, and these lines were screened for the production of anticolon antibodies. B-cell lines were then cloned by limiting dilution culture, and messenger RNA expression of immunoglobulin heavy-chain variable region (V_H) was assessed. Results: V_H gene families used in B-cell lines established from LPLs of normal controls were diverse, and B-cell lines from UC LPLs expressed a restricted V_H3 family usage. All 15 clones from UC used a restricted V_H3 gene family, whereas diverse V_H gene families were used by 24 clones from normal controls. The analysis of nucleotide sequences indicated that these clones were derived from various germline gene segments. Conclusions: The restricted V_H gene usage in anticolon autoantibodies producing B-cell clones suggests that a particular antigenic stimulus contributes to the pathogenesis of UC.

クローン病におけるIL-18の関与

日比紀文，金井隆典，渡辺守，岡沢啓，佐藤俊朗

日本臨床免疫学会会誌 23 （ 6 ） 607-610 2000年12月

研究論文（その他学術会議資料等）, 共著

実験動物モデル

佐藤俊朗，渡辺守，日比紀文

消化器病セミナー 77   111-122 1999年12月

研究論文（学術雑誌）, 共著