Junichi Matsuo1, Shunichi Kimura2, Akihiro Yamamura2, Cai Ping Koh1, Md Zakir Hossain1, Dede Liana Heng1, Kazuyoshi Kohu1, Dominic Chih-Cheng Voon1, Hiroshi Hiai3, Michiaki Unno4, Jimmy Bok Yan So5, Feng Zhu6, Supriya Srivastava1, Ming Teh7, Khay Guan Yeoh8, Motomi Osato9, Yoshiaki Ito10. 1. Cancer Science Institute of Singapore, National University of Singapore, Singapore. 2. Cancer Science Institute of Singapore, National University of Singapore, Singapore; Department of Surgery, Graduate School of Medicine, Tohoku University, Miyagi, Japan. 3. Kyoto Disease Model Institute, Kyoto Science and Technology Center, Kyoto, Japan. 4. Department of Surgery, Graduate School of Medicine, Tohoku University, Miyagi, Japan. 5. Department of Surgery, Yong Loo Lin School of Medicine, National University of Singapore, Singapore. 6. Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, Singapore. 7. Department of Pathology, National University Health System, Singapore. 8. Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, Singapore; Department of Gastroenterology and Hepatology, National University Health System, Singapore. 9. Cancer Science Institute of Singapore, National University of Singapore, Singapore; Institute of Bioengineering and Nanotechnology, A*STAR, Singapore; Department of Pediatrics, Yong Loo Lin School of Medicine, National University of Singapore, Singapore; International Research Center for Medical Sciences, Kumamoto University, Kumamoto, Japan. Electronic address: csimo@nus.edu.sg. 10. Cancer Science Institute of Singapore, National University of Singapore, Singapore; Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, Singapore. Electronic address: csiitoy@nus.edu.sg.
Abstract
BACKGROUND & AIMS: Little is known about the mechanisms of gastric carcinogenesis, partly because it has been a challenge to identify characterize gastric stem cells. Runx genes regulate development and their products are transcription factors associated with cancer development. A Runx1 enhancer element, eR1, is a marker of hematopoietic stem cells. We studied expression from eR1 in the stomach and the roles of gastric stem cells in gastric carcinogenesis in transgenic mice. METHODS: We used in situ hybridization and immunofluorescence analyses to study expression of Runx1 in gastric tissues from C57BL/6 (control) mice. We then created mice that expressed enhanced green fluorescent protein (EGFP) or CreERT2 under the control of eR1 (eR1-CreERT2;Rosa-Lox-Stop-Lox [LSL]-tdTomato, eR1-CreERT2;Rosa-LSL-EYFP mice). Gastric tissues were collected and lineage-tracing experiments were performed. Gastric organoids were cultured from eR1-CreERT2(5-2);Rosa-LSL-tdTomato mice and immunofluorescence analyses were performed. We investigated the effects of expressing oncogenic mutations in stem cells under control of eR1 using eR1-CreERT2;LSL-KrasG12D/+ mice; gastric tissues were collected and analyzed by histology and immunofluorescence. RESULTS: Most proliferation occurred in the isthmus; 86% of proliferating cells were RUNX1-positive and 76% were MUC5AC-positive. In eR1-EGFP mice, EGFP signals were detected mainly in the upper part of the gastric unit, and 83% of EGFP-positive cells were located in the isthmus/pit region. We found that eR1 marked undifferentiated stem cells in the isthmus and a smaller number of terminally differentiated chief cells at the base. eR1 also marked cells in the pyloric gland in the antrum. Lineage-tracing experiments demonstrated that stem cells in the isthmus and antrum continuously gave rise to mature cells to maintain the gastric unit. eR1-positive cells in the isthmus and pyloric gland generated organoid cultures in vitro. In eR1-CreERT2;LSL-Kras G12D/+ mice, MUC5AC-positive cells rapidly differentiated from stem cells in the isthmus, resulting in distinct metaplastic lesions similar to that observed in human gastric atrophy. CONCLUSIONS: Using lineage-tracing experiments in mice, we found that a Runx1 enhancer element, eR1, promotes its expression in the isthmus stem cells of stomach corpus as well as pyloric gland in the antrum. We were able to use eR1 to express oncogenic mutations in gastric stem cells, proving a new model for studies of gastric carcinogenesis.
BACKGROUND & AIMS: Little is known about the mechanisms of gastric carcinogenesis, partly because it has been a challenge to identify characterize gastric stem cells. Runx genes regulate development and their products are transcription factors associated with cancer development. A Runx1 enhancer element, eR1, is a marker of hematopoietic stem cells. We studied expression from eR1 in the stomach and the roles of gastric stem cells in gastric carcinogenesis in transgenic mice. METHODS: We used in situ hybridization and immunofluorescence analyses to study expression of Runx1 in gastric tissues from C57BL/6 (control) mice. We then created mice that expressed enhanced green fluorescent protein (EGFP) or CreERT2 under the control of eR1 (eR1-CreERT2;Rosa-Lox-Stop-Lox [LSL]-tdTomato, eR1-CreERT2;Rosa-LSL-EYFP mice). Gastric tissues were collected and lineage-tracing experiments were performed. Gastric organoids were cultured from eR1-CreERT2(5-2);Rosa-LSL-tdTomato mice and immunofluorescence analyses were performed. We investigated the effects of expressing oncogenic mutations in stem cells under control of eR1 using eR1-CreERT2;LSL-KrasG12D/+ mice; gastric tissues were collected and analyzed by histology and immunofluorescence. RESULTS: Most proliferation occurred in the isthmus; 86% of proliferating cells were RUNX1-positive and 76% were MUC5AC-positive. In eR1-EGFP mice, EGFP signals were detected mainly in the upper part of the gastric unit, and 83% of EGFP-positive cells were located in the isthmus/pit region. We found that eR1 marked undifferentiated stem cells in the isthmus and a smaller number of terminally differentiated chief cells at the base. eR1 also marked cells in the pyloric gland in the antrum. Lineage-tracing experiments demonstrated that stem cells in the isthmus and antrum continuously gave rise to mature cells to maintain the gastric unit. eR1-positive cells in the isthmus and pyloric gland generated organoid cultures in vitro. In eR1-CreERT2;LSL-Kras G12D/+ mice, MUC5AC-positive cells rapidly differentiated from stem cells in the isthmus, resulting in distinct metaplastic lesions similar to that observed in humangastric atrophy. CONCLUSIONS: Using lineage-tracing experiments in mice, we found that a Runx1 enhancer element, eR1, promotes its expression in the isthmus stem cells of stomach corpus as well as pyloric gland in the antrum. We were able to use eR1 to express oncogenic mutations in gastric stem cells, proving a new model for studies of gastric carcinogenesis.
Authors: Elise S Demitrack; Gail B Gifford; Theresa M Keeley; Nobukatsu Horita; Andrea Todisco; D Kim Turgeon; Christian W Siebel; Linda C Samuelson Journal: Am J Physiol Gastrointest Liver Physiol Date: 2016-12-08 Impact factor: 4.052
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