Identification of Stem Cells in the Epithelium of the Stomach Corpus and Antrum of Mice.
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.



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.



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.



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.
Cancer Science Institute of Singapore, National University of Singapore, Singapore.
Junichi
J
Matsuo
Cancer Science Institute of Singapore, National University of Singapore, Singapore; Department of Surgery, Graduate School of Medicine, Tohoku University, Miyagi, Japan.
Shunichi
S
Kimura
Cancer Science Institute of Singapore, National University of Singapore, Singapore; Department of Surgery, Graduate School of Medicine, Tohoku University, Miyagi, Japan.
Akihiro
A
Yamamura
Cancer Science Institute of Singapore, National University of Singapore, Singapore.
Cai Ping
CP
Koh
Cancer Science Institute of Singapore, National University of Singapore, Singapore.
Md Zakir
MZ
Hossain
Cancer Science Institute of Singapore, National University of Singapore, Singapore.
Dede Liana
DL
Heng
Cancer Science Institute of Singapore, National University of Singapore, Singapore.
Kazuyoshi
K
Kohu
Cancer Science Institute of Singapore, National University of Singapore, Singapore.
Dominic Chih-Cheng
DC
Voon
Kyoto Disease Model Institute, Kyoto Science and Technology Center, Kyoto, Japan.
Hiroshi
H
Hiai
Department of Surgery, Graduate School of Medicine, Tohoku University, Miyagi, Japan.
Michiaki
M
Unno
Department of Surgery, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.
Jimmy Bok Yan
JB
So
Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.
Feng
F
Zhu
Cancer Science Institute of Singapore, National University of Singapore, Singapore.
Supriya
S
Srivastava
Department of Pathology, National University Health System, Singapore.
Ming
M
Teh
Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, Singapore; Department of Gastroenterology and Hepatology, National University Health System, Singapore.
Khay Guan
KG
Yeoh
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.
Motomi
M
Osato
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.
Yoshiaki
Y
Ito
D000818:Animals; D063646:Carcinogenesis; D002454:Cell Differentiation; D019070:Cell Lineage; D049109:Cell Proliferation; D050676:Core Binding Factor Alpha 2 Subunit / Q000235:genetics* / Q000378:metabolism; D004742:Enhancer Elements, Genetic / Q000235:genetics*; D005753:Gastric Mucosa / Q000378:metabolism* / Q000473:pathology*; D015870:Gene Expression; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D019394:Ki-67 Antigen / Q000378:metabolism; D008679:Metaplasia / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D055271:Mucin 5AC / Q000378:metabolism; D011706:Pyloric Antrum / Q000378:metabolism / Q000473:pathology*; D012333:RNA, Messenger / Q000378:metabolism*; D013234:Stem Cells / Q000378:metabolism* / Q000473:pathology*; D013274:Stomach Neoplasms / Q000235:genetics* / Q000473:pathology*; D046509:Tissue Culture Techniques
D050676:Core Binding Factor Alpha 2 Subunit; D019394:Ki-67 Antigen; C524679:Muc5ac protein, mouse; D055271:Mucin 5AC; D012333:RNA, Messenger; C493726:Runx1 protein, mouse; D049452:Green Fluorescent Proteins
Cancer Stem Cell; Mouse Model; Oncogene; Stomach Cancer