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      Modeling colorectal tumorigenesis using the organoids derived from conditionally immortalized mouse intestinal crypt cells (ciMICs)

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          Abstract

          Intestinal cancers are developed from intestinal epithelial stem cells (ISCs) in intestinal crypts through a multi-step process involved in genetic mutations of oncogenes and tumor suppressor genes. ISCs play a key role in maintaining the homeostasis of gut epithelium. In 2009, Sato et al established a three-dimensional culture system, which mimicked the niche microenvironment by employing the niche factors, and successfully grew crypt ISCs into organoids or Mini-guts in vitro. Since then, the intestinal organoid technology has been used to delineate cellular signaling in ISC biology. However, the cultured organoids consist of heterogeneous cell populations, and it was technically challenging to introduce genomic changes into three-dimensional organoids. Thus, there was a technical necessity to develop a two-dimensional ISC culture system for effective genomic manipulations. In this study, we established a conditionally immortalized mouse intestinal crypt (ciMIC) cell line by using a piggyBac transposon-based SV40 T antigen expression system. We showed that the ciMICs maintained long-term proliferative activity under two-dimensional niche factor-containing culture condition, retained the biological characteristics of intestinal epithelial stem cells, and could form intestinal organoids in three-dimensional culture. While in vivo cell implantation tests indicated that the ciMICs were non-tumorigenic, the ciMICs overexpressing oncogenic β-catenin and/or KRAS exhibited high proliferative activity and developed intestinal adenoma-like pathological features in vivo. Collectively, these findings strongly suggested that the engineered ciMICs should be used as a valuable tool cell line to dissect the genetic and/or epigenetic underpinnings of intestinal tumorigenesis.

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          Most cited references50

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

          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 cryptvillus organoids. Tracing experiments indicate that the Lgr5(+) stem-cell hierarchy is maintained in organoids. We conclude that intestinal cryptvillus 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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            Cancer genome landscapes.

            Over the past decade, comprehensive sequencing efforts have revealed the genomic landscapes of common forms of human cancer. For most cancer types, this landscape consists of a small number of "mountains" (genes altered in a high percentage of tumors) and a much larger number of "hills" (genes altered infrequently). To date, these studies have revealed ~140 genes that, when altered by intragenic mutations, can promote or "drive" tumorigenesis. A typical tumor contains two to eight of these "driver gene" mutations; the remaining mutations are passengers that confer no selective growth advantage. Driver genes can be classified into 12 signaling pathways that regulate three core cellular processes: cell fate, cell survival, and genome maintenance. A better understanding of these pathways is one of the most pressing needs in basic cancer research. Even now, however, our knowledge of cancer genomes is sufficient to guide the development of more effective approaches for reducing cancer morbidity and mortality.
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              Genome editing. The new frontier of genome engineering with CRISPR-Cas9.

              The advent of facile genome engineering using the bacterial RNA-guided CRISPR-Cas9 system in animals and plants is transforming biology. We review the history of CRISPR (clustered regularly interspaced palindromic repeat) biology from its initial discovery through the elucidation of the CRISPR-Cas9 enzyme mechanism, which has set the stage for remarkable developments using this technology to modify, regulate, or mark genomic loci in a wide variety of cells and organisms from all three domains of life. These results highlight a new era in which genomic manipulation is no longer a bottleneck to experiments, paving the way toward fundamental discoveries in biology, with applications in all branches of biotechnology, as well as strategies for human therapeutics. Copyright © 2014, American Association for the Advancement of Science.
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                Author and article information

                Contributors
                Journal
                Genes Dis
                Genes Dis
                Genes & Diseases
                Chongqing Medical University
                2352-4820
                2352-3042
                28 January 2021
                November 2021
                28 January 2021
                : 8
                : 6
                : 814-826
                Affiliations
                [a ]Departments of Gastrointestinal Surgery, Medicine/Gastroenterology, and Obstetrics and Gynecology, The First Affiliated Hospital of Chongqing Medical University, Chongqing, 400016, PR China
                [b ]Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 606037, USA
                [c ]Department of Oncology, The PLA Rocket Force Characteristic Medical Center, Beijing, 100088, PR China
                [d ]Departments of Burn & Plastic Surgery, West China Hospital of Sichuan University, Chengdu, Sichuan Province, 610041, PR China
                [e ]Department of Orthopaedic Surgery, The First Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou, Guangdong Province, 510405, PR China
                [f ]Departments of Orthopaedic Surgery, The Affiliated Zhongnan Hospital of Wuhan University, Wuhan, Hubei Province, 430071, PR China
                [g ]Department of Orthopaedic Surgery, The Affiliated Hospital of Qingdao University, Qingdao, Shandong Province, 266000, PR China
                [h ]Department of Clinical Laboratory Medicine, Chongqing General Hospital Affiliated with the University of Chinese Academy of Sciences, Chongqing, 400013, PR China
                [i ]Ministry of Education Key Laboratory of Diagnostic Medicine, and the School of Laboratory Diagnostic Medicine, Chongqing Medical University, Chongqing, 400016, PR China
                [j ]Department of Orthopaedics, Union Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei Province, 430022, PR China
                [k ]Department of Spine Surgery, Second Xiangya Hospital, Central South University, Changsha, Hunan Province, 410008, PR China
                [l ]Department of Oncology and Southwest Cancer Center, Southwest Hospital, Army Medical University, Chongqing, 400038, PR China
                Author notes
                []Corresponding author. Department of Oncology, The PLA Rocket Force Characteristic Medical Center, #16 Xinjiekouwai Avenue, Xicheng District, Beijing, 100088, China. quiet_wang@ 123456163.com
                [∗∗ ]Corresponding author. Department of Gastroenterology, The First Affiliated Hospital, Chongqing Medical University, #1 Youyi Road, Yuzhong District, Chongqing, 400016, China. zhbingqiang@ 123456163.com
                Article
                S2352-3042(21)00011-8
                10.1016/j.gendis.2021.01.004
                8427244
                34522710
                40a92545-2e1f-4973-8c7d-4219ae49b76a
                © 2021 Chongqing Medical University. Production and hosting by Elsevier B.V.

                This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

                History
                : 7 July 2020
                : 18 December 2020
                : 20 January 2021
                Categories
                Full Length Article

                cancer modeling,conditional immortalization,mini-gut organoids,mouse intestinal crypt (mic) cells,tumorigenesis

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