In vitro differentiation of Runx3-/- p53-/- gastric epithelial cells into intestinal type cells

Abstract

We have reported that a lack of RUNX3 function is causally associated with gastric carcinogenesis. We have also presented evidence that loss of Runx3 may be related to the genesis of intestinal metaplasia because expression of RUNX3 is reduced in some intestinal metaplasias, and some Runx3(-/-)p53(-/-) gastric epithelial cells differentiate into intestinal type cells in vivo. Recently several reports have indicated that blood cells play important roles in the gastric carcinogenesis. In the present study, we therefore examined whether Runx3(-/-)p53(-/-) gastric epithelial cells differentiate autonomously into intestinal type cells, or whether the presence of other cells is necessary for the differentiation in vitro. When Runx3(-/-)p53(-/-) gastric epithelial cells were cultured with collagen gels, they did not exhibit any morphogenesis and differentiated poorly. When cultured with fetal mouse gastric mesenchymes, the cells formed glandular structures and differentiated into surface mucous cells, but differentiation of intestinal type cells was never observed. When cultured with Matrigel, the cells formed glandular structures, and some cells differentiated into intestinal type cells in vitro. Reverse transcription-polymerase chain reaction analysis showed that the cells expressed stomach-specific genes, and their levels decreased gradually during the culture. The cells expressed some intestine-specific genes weakly at the start of culture, and their levels were increased with time in culture. We therefore conclude that Runx3(-/-)p53(-/-) gastric epithelial cells differentiate into intestinal type cells in combination with Matrigel in the absence of other cell types. Extracellular matrix, not blood cells, may play a role in the genesis of intestinal metaplasia.

Figure 1

The method of organ culture. GIF‐11 cells were cultured on Millipore filters covered with a drop of collagen gel or Matrigel. The next day, the cells were covered with fetal mouse gastric mesenchymes or a drop of collagen gel or Matigel, and the cells with mesenchymal components were laid on a stainless steel grid in dishes, and were cultured at the air–liquid interface for up to 6 weeks.

Figure 2

Phase contast micrographs of GIF‐11 cells cultured on (a) plastic, (b) collagen gel, and (c) Matrigel for 1 day. (b) Some cells on the collagen gel attach closely to each other to form small cell masses (arrow), (c) whereas on Matrigel, much greater cell masses are formed. Scale bars = 100 µm.

Figure 3

Morphogenesis and differentiation in vitro of GIF‐11 cells cultured with (a–c) collagen gels, (d–f) gastric mesenchymes, and (g–i) Matrigel, for (a,d,g) 1, (b,e,h) 2, and (c,f,i) 3 weeks. Sections are stained with periodic acid–Schiff (PAS) and hematoxylin. (f) Closed arrows show surface mucous cells characterized by large PAS‐positive materials and elongated nuclei. Some fibroblasts die during long‐term culture, and open arrows in (f) show the nuclei of dying cells. No leukocytes can be found in the mesenchyme in organ culture. Scale bars = 100 µm.

Figure 4

Differentiation of mucus‐containing cells in an adult mouse (a,c,e) stomach and (b,d,f) small intestine, and in GIF‐11 cells cultured with Matrigel for (g–i) 4 and (j–l) 6 weeks. Periodic acid–Schiff (PAS) and hematoxylin staining shows that (g,j) some GIF‐11 cells contain large PAS‐positive mucus droplets (arrows), (b) found specifically in goblet cells in the intestine (arrows). Alcian blue (AB)‐neutral red and high iron diamine (HID)‐AB staining shows that (h,k) the mucus droplets in GIF‐11 cells are AB‐positive (arrows) and (i,l) HID‐positive (arrows), (d,f) as is the case for mucus in goblet cells, (c,e) whereas mucus in gastric epithelial cells is negative for both stains. Scale bars = 50 µm.

Figure 5

Reverse transcription–polymerase chain reaction analysis on the differentiation of GIF‐11 cells combined with collagen gel (middle panel) or Matrigel (right panel) for 1 day (1 d), 2 weeks (2 w), 4 weeks (4 w), and 6 weeks (6 w). Mucin1 (Muc1), Sox2, intrinsic factor (IF), Mucin2 (Muc2), Cdx2, and sucrase isomaltase (SI) are used as tissue‐specific markers for differentiation, as Muc1, Sox2, and IF are expressed explicitly by gastric (stomach) epithelial cells, and Muc2, Cdx2, and SI are expressed specifically by intestinal (duodenum) epithelial cells (left panel). Gapdh, glyceraldehyde 3‐phosphate dehydrogenase.