Details of a Researcher - Sato, Toshiro

Papers - Sato, Toshiro

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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 2019.03

© 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 2019.02

ISSN 00165085

© 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 2018.12

ISSN 19345909

© 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 Yoshimasa, Nakaoka Toshiaki, Muramatsu Toshihide, Ojima Hidenori, Sukeda Aoi, Sugiyama Yuko, Uchida Ryoei, Furukawa Ryo, Kitahara Aya, Sato Toshiro, Kanai Yae, Saito Hidetsugu

Scientific Reports (SPRINGER NATURE) 8 ( 1 ) 2018.12

Research paper (scientific journal), Joint Work, Accepted, ISSN 2045-2322

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 Kosaku, Toshimitsu Kohta, Takano Ai, Fujii Masayuki, Shimokawa Mariko, Ohta Yuki, Matano Mami, Seino Takashi, Nishikori Shingo, Ishikawa Keiko, Kawasaki Kenta, Togasaki Kazuhiro, Takahashi Sirirat, Sukawa Yasutaka, Ishida Hiroki, Sugimoto Shinya, Kawakubo Hirofumi, Kim Jihoon, Kitagawa Yuko, Sekine Shigeki, Koo Bon Kyoung, Kanai Takanori, Sato Toshiro

Cell (Elsevier) 174 ( 4 ) 856 - 869.e17 2018.08

Research paper (scientific journal), Joint Work, Accepted, ISSN 0092-8674

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 Cindy J.M., Williams Giuliani Nerys, Balak Jeetindra, Ringnalda Femke, van Gurp Léon, Huch Meritxell, Boj Sylvia F., Sato Toshiro, Kester Lennart, de Sousa Lopes Susana M.Chuva, Roost Matthias S., Bonner-Weir Susan, Engelse Marten A., Rabelink Ton J., Heimberg Harry, Vries Robert G.J., van Oudenaarden Alexander, Carlotti Françoise, Clevers Hans, de Koning Eelco J.P.

Stem Cell Reports (Stem Cell Reports) 10 ( 3 ) 1088 - 1101 2018.03

Research paper (scientific journal), Joint Work, Accepted, ISSN 2213-6711

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 ) 2018.02

© 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 Shinya, Ohta Yuki, Fujii Masayuki, Matano Mami, Shimokawa Mariko, Nanki Kosaku, Date Shoichi, Nishikori Shingo, Nakazato Yoshihiro, Nakamura Tetsuya, Kanai Takanori, Sato Toshiro

Cell Stem Cell (Elsevier) 22 ( 2 ) 171 - 176.e5 2018.02

Research paper (scientific journal), Joint Work, ISSN 1934-5909

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 Takashi, Sakakibara Sayuri, Jinnohara Toshi, Hachisuka Masami, Tachibana Naoko, Hidano Shinya, Kobayashi Takashi, Kimura Shunsuke, Iwanaga Toshihiko, Nakagawa Tomoo, Katsuno Tatsuro, Kato Naoya, Akiyama Taishin, Sato Toshiro, Williams Ifor R., Ohno Hiroshi

Journal of Experimental Medicine (Rockefeller University Press) 215 ( 2 ) 501 - 519 2018.02

Research paper (scientific journal), Joint Work, Accepted, ISSN 0022-1007

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 Akiko, Nakano Yukiko, Saitoh Takako, Gouraud Sabine S.S., Yamaguchi Yoshiki, Sato Toshiro, Sasaki Nobuo, Kojima-Aikawa Kyoko

Glycobiology (Oxford University Press) 28 ( 1 ) 21 - 31 2018.01

Research paper (scientific journal), Joint Work, Accepted, ISSN 0959-6658

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 Tetsuya, Sato Toshiro

CMGH (Elsevier) 5 ( 1 ) 51 - 60 2018.01

Research paper (scientific journal), Joint Work, Accepted

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 Takashi, Kawasaki Shintaro, Shimokawa Mariko, Tamagawa Hiroki, Toshimitsu Kohta, Fujii Masayuki, Ohta Yuki, Matano Mami, Nanki Kosaku, Kawasaki Kenta, Takahashi Sirirat, Sugimoto Shinya, Iwasaki Eisuke, Takagi Junichi, Itoi Takao, Kitago Minoru, Kitagawa Yuko, Kanai Takanori, Sato Toshiro

Cell Stem Cell (Elsevier) 22 ( 3 ) 454 - 467 2018.01

Research paper (scientific journal), Accepted, ISSN 1934-5909

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 2017.08

Research paper (scientific journal), Joint Work, ISSN 2211-1247

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 ) 2017.07

Research paper (scientific journal), Joint Work, ISSN 1756-994X

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Establishment of 3D intestinal organoid cultures from intestinal stem cells

Sugimoto Shinya, Sato Toshiro

Methods in Molecular Biology (Springer) 1612 97 - 105 2017.06

Research paper (scientific journal), Joint Work, Accepted

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.

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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 2017.06

Research paper (scientific journal), Joint Work, ISSN 0022-1007

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 2017.05

Research paper (scientific journal), Joint Work, ISSN 0028-0836

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 2017.05

Research paper (scientific journal), Joint Work, ISSN 1465-7392

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 2017.04

Joint Work

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.

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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 ) 2016.10

Research paper (scientific journal), ISSN 1553-7390

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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