Isolation and characterization of human gastric cell lines with stem cell phenotypes.
OBJECTIVE
The aim of this study was to develop an in vitro human gastric stem and/or progenitor cell model that may be used to study the mechanism of gastric carcinogenesis induced by Helicobacter pylori infection.


METHODS
Human gastric biopsy was minced and digested with collagenase and dispase and cultured in a low-calcium medium (serum-free keratinocyte medium; keratinocyte-SFM) supplemented with N-acetyl-L-cysteine and L-ascorbic acid 2-phosphate. Actively proliferating epithelial colonies with sustained growth were isolated and characterized for karyotype and phenotypes related to stem cell characteristics including proliferation and differentiation potential, ability of anchorage-independent growth (AIG), gap junctional intercellular communication (GJIC) and the expression of Oct-4, a transcription factor previously shown to be expressed in embryonic stem cells, adult stem cells and undifferentiated tumor cells. To study the carcinogenic effect of H. pylori infection, gastric stem and/or progenitor cells were incubated with H. pylori culture products and/or N-methyl-N-nitro-N-nitrosoguanidine (MNNG), a chemical carcinogen, to see the telomerase activation.


RESULTS
Multiple cell lines with stem cell features were isolated by this new cell culture method. The results based on detailed characterization of one cell clone, KMU-GI2, revealed stem cell features of these cells. The initial clone contained mostly undifferentiated epithelial-like cells, which, upon subculture and propagation, gave rise to a heterogeneous cell population. Single cell-derived subclones, similar to the parental population, retained high differentiation potential and were capable of giving rise to many morphologically different cell types (i.e. epithelial-like, glial or neuron-like, round and various peculiar-shaped cells). Although these cells were normal in karyotype and competent in GJIC, they had the ability to grow in soft agar. Cells expressing epithelial membrane antigen (EMA), mucin 5AC, glial fibrillary acidic protein (GFAP), cytokeratin-18 (CK-18), trefoil factor 1 (TFF-1) and Oct-4 were found in the cell culture, but not E-cadherin-, gastrin- or telomerase-expressing cells. Furthermore, spontaneously immortalized non-tumorigenic clones could be derived from the cell population. After treating these cell cultures with the chemical carcinogen, MNNG and H. pylori culture products for 5 days, telomerase activity and telomerase mRNA expression were significantly elevated, while treatment with either of them showed no effect.


CONCLUSIONS
The new cell culture method can be used to develop gastric epithelial cell clones with sustained growth from endoscopic biopsy. The gastric cell clone showed several stem and/or progenitor cell phenotypes (i.e. the ability of AIG, high differentiation capacity, high susceptibility to spontaneous immortalization and the expression of Oct-4). The telomerase expression in these gastric stem and/or progenitor cells can be upregulated by exposure to H. pylori culture products and MNNG, an important step in neoplastic transformation. These results show that putative human gastric stem and/or progenitor cell clones can be developed by our method and these cells could be useful for studying the mechanisms of human gastric carcinogenesis including the mechanism of action of H. pylori, as well as the regulation of the proliferation and differentiation of human gastric mucosa.
Yang
Yuan-Chieh
YC
Department of Laboratory Medicine, and Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan.
Wang
Sheng-Wen
SW
Hung
Hsiao-Yen
HY
Chang
Chia-Cheng
CC
Wu
I-Chen
IC
Huang
Yeou-Lih
YL
Lin
Tsai-Ming
TM
Tsai
Jin-Lian
JL
Chen
Angela
A
Kuo
Fu-Chen
FC
Wang
Wen-Ming
WM
Wu
Deng-Chyang
DC
D001706:Biopsy; D002450:Cell Communication; D018929:Cell Culture Techniques; D002455:Cell Division; D002460:Cell Line; D017629:Gap Junctions / Q000502:physiology; D005753:Gastric Mucosa / Q000166:cytology* / Q000502:physiology; D016480:Helicobacter pylori / Q000502:physiology; D006801:Humans; D007621:Karyotyping; D007700:Kinetics; D013234:Stem Cells / Q000166:cytology* / Q000382:microbiology / Q000502:physiology; D013270:Stomach / Q000166:cytology* / Q000382:microbiology / Q000502:physiology; D013274:Stomach Neoplasms / Q000382:microbiology; D014157:Transcription Factors / Q000235:genetics
D014157:Transcription Factors