Establishment of a gastric epithelial progenitor cell line from a transgenic mouse expressing the simian virus 40 large T antigen gene in the parietal cell lineage

Abstract

Objective: In this study the gastric mucosa of transgenic mice expressing the simian virus 40 large T antigen gene in the parietal cell lineage is used to establish and characterize a new epithelial progenitor cell line. In these mice, proliferation and amplification of preparietal cells preclude their maturation into acid-secreting parietal cells leading to achlorohydria, hyperplasia, dysplasia and eventually gastric adenocarcinoma.

Materials and methods: Enzymatically dispersed gastric epithelial cells were cultured, cloned and screened using immunohistochemical methods, for expression of a variety of biomarkers of differentiated pit, parietal, enteroendocrine and neck/zymogenic cells.

Results: A biomarker-deficient cell line whose ultrastructural features resembled those of mouse gastric epithelial progenitor cells was established. Treatment with either hydrocortisone or oestrogen significantly enhanced proliferation of these cells, whereas retinoic acid inhibited their growth. No change in differentiation was detected with any of these treatments; however, when these cells were injected subcutaneously into nude mice, they proliferated to form tumours and undergo partial differentiation towards parietal cell lineage.

Conclusion: This mouse gastric epithelial progenitor cell line could be useful as an in vitro model to study growth properties, proliferation and differentiation of a subpopulation of gastric epithelial progenitor cells and also to study gastric carcinogenesis.

Figure 1

Schematic representation of the normal gastric gland and changes that occur in its isthmal progenitor cells (between the two arrows at the left side) and their descendent cell lineages, during expression of the SV40 large T antigen, in the parietal cell lineage (red). Note amplification of the progenitors of the parietal cell lineage and their transdifferentiation into invasive pre‐enteroendocrine cells (yellow). Pit and zymogenic cell lineages appear green and purple, respectively.

Figure 2

Establishment of the mouse gastric epithelial progenitor (mGEP) cell line. Light micrographs showing attached single epithelial‐like cell (a), a multinucleate cell (b), small epithelial‐like colony (c) and confluent monolayer (d) as they progressively evolve during the first month after dispersion and plating of the transgenic gastric mucosal cells. Scanning electron micrograph showing attached, tightly packed cells (e), transmission electron micrograph of the cells showing distinct epithelial junction at the arrow (f). ×400 (a, b); ×200 (c, d); ×800 (e); ×6000 (f) original magnifications.

Figure 3

Immunohistochemical analysis of the developing mGEP cell line using phalloidin (a), antibodies specific for actin (b) and cytokeratin (c). (a) The two initially attached cell types are stained to reveal actin, but the cell on the left appears typically mesenchymal and the right one appears epithelial‐like. (b) Confluent layer of the mGEP cell line stained positive for actin. (c) Monolayer of mGEP cells showing cytoplasmic expression of cytokeratin. ×400 (a); ×200 (b); ×300 (c) original magnifications.

Figure 4

Electron micrographs of the immortalized mouse gastric epithelial progenitor (mGEP) cells showing their ultrastructural features. (a) Note progenitor cell‐like features: large nucleus in relation to cytoplasm, much diffuse chromatin, and many free ribosomes. In addition, some microvilli (arrows) cut at different orientations are projecting from the apical surface. (b) Note the few small secretory granules; they appear electron dense and round or ovoid similar to those of pre‐enteroendocrine cells. (c) The apical cytoplasm of mGEP cell transplanted into a nude mouse showing numerous tubulovesicular elements characteristic of parietal cell lineage; few scattered electron dense granules are also present. (d) Transplanted mGEP cell showing a small group of electron dense granules characteristic of pre‐enteroendocrine cells. ×6000 (a, b, d); ×11 000 (c) original magnifications.

Figure 5

Effects of hydrocortisone, retinoic acid and oestrogen on the expansion of cells of the mouse gastric epithelial progenitor (mGEP) line. Cells were grown in serum‐free medium containing 0.5, 1 or 2 nm of hydrocortisone, oestrogen or retinoic acid. Control cells received serum‐free medium with no hormone. After 1 day, cells were trypsinized and quantified using a haemocytometer. Each bar shows data from triplicate wells presented as mean ± SEM.