Isolation and characterization of stomach stem cell

Northern blot and immunohistochemical analysis of Vsig1 demonstrate that Vsig1 gene expression begins around E13.5 of stomach development. VSIG1 protein is detected in all cells of pseudostratified epithelium in that stage. There was no expression in mesenchymal layer. This highly specific expression in early stage of stomach development gives us a suggestion that Vsig1 gene is expressed in stomach stem cells.

As described in above section, the stomach epithelium begins to differentiate in E13.5 and form two distinct types of epithelium. Stratified squamous epithelium is in anterior region and monolayered glandular epithelium is in posterior region. Even differentiation of specific cell lineages and invaginations of epithelium to form glands

begin already in the embryo, but mature glands are not formed before birth. At birth, 90% of the cells in the rudimentary gastric units are precursor cells. The percentage of progenitor cells is reduced to 20% in the first 7 days postnatally (P1–P7), when cellular differentiation occurs (Karam and Gordon, 1997).

Existing reports on the localization of epithelial stem cells in gastric unit are somewhat controversial. Karam and Leblond (1992) combined radioactive thymidin labelling and electron microscopy analysis to identify cell types in the gastric unit of the glandular region of adult mice. They found out that the multipotent stem cells of the glandular epithelium are localized in the isthmus region. They also identified three progenitor cell lineages, pre-pit, pure-neck and pre-parietal precursors, which become differentiated from multipotent stem cells. It is also considered that the antral gastric mucosa, including the endocrine cells derived from a common stem cell located in the isthmus region (Karam and Leblond, 1995). Several studies have shown that stomach glands are homotypic, and thus, all of the different cells in one gland are clonally derived from the same parent progenitor cell (Thompson et al., 1990; Tatematsu et all., 1994). Other studies have shown that in the beginning of invagination, most glands are initially polyclonal with three or four stem cells per gland, and they become monoclonal during the first six weeks of murine life (Nomura et al., 1998).

Gastric stem or progenitor cells have not been well-characterized due to the lack of specific markers that permit their prospective recognition. Recently, Barker et al (2007) have used the cDNA probe of LGR5, which is an intestine stem cell marker, to localize the epithelial stem cells in gastric unit. The in-situ hybridization showed that epithelial stem cells are localized on the base of gastric unit similar to localization of epithelial stem cells in the base of intestinal crypt (Barker et al., 2007).

The high expression of Vsig1 in glandular epithelium of embryonic stomach, which mainly contains epithelial stem and progenitor cells, suggest that VSIG1 could be useful as marker for isolation of the gastric epithelial stem cells. To isolate and characterize the epithelial stem cells of stomach, we have generated Vsig1-EGFP transgenic mice.

Immunohistological studies revealed that the transgenic EGFP allele is expressed in most cells of glandular epithelium of stomach from embryos at E18.5. In adult stomach, no GFP-positive cells were detected in most gastric units. Approximately, every 20 gastric unit has only one unit which has one or two GFP-positive cells. This result demonstrates that the expression pattern of transgenic Vsig1-EGFP in embryonic stomach is similar to that of endogenous Vsig1 gene. In contrast, the expression pattern

of transgenic allele in adult stomach is different to that of endogenous Vsig1 gene. It can be assumed that the 1.2-kb long 3’-untranslated region of Vsig1 gene contains elements, which are responsible for stability of Vsig1 mRNA in glandular epithelial of adult stomach. Such sequences are lacking in transgenic EGFP-mRNA. The results of many studies showed that the 3’-untranslated region of a gene influence the stability of its mRNA (Kakok et al., 2004; Ross, 1995; Adham et al., 2008). Shirneshan et al (2008) have generated a transgenic mouse Insl3-Ins, in which the human insulin gene is under the control of Leydig-cell specific gene (Insl3). They found that the expression of transgenic insulin mRNA was very low in Leydig cells. Replacement of 3’-untranslated region of insulin with that of Insl3 in the second transgenic allele resulted in a significantly higher transgene expression. These results suggest that the 3’-untranslated region of Insl3 contains elements, which are responsible for stability of transgenic transcript in Leydig cells (Shirneshan et al., 2008).

Alternative explanation came from our recent Northern blot analysis showing that Vsig1 gene transcribes 2.7-kb and 2.0-kb mRNA isoforms in adult stomach. Both transcripts are different in the length of the 3’-untranslated regions and in the expression level. The 2.7-kb mRNA is highly expressed in adult stomach and contains 1.5-kb 3’-UTR, while the 2.0-kb mRNA is lowly expressed in adult stomach and contains 800-bp 3’-UTR, which is spliced at the first polyadenylation signal of the gene. Low expression level of the 2.0-kb transcript in adult stomach suggest that this transcript is restricted to the epithelial stem cells similar to the transcript of the transgenic Vsig1—EGFP. Similar results were also observed from the characterization of the Villin-LacZ and Villin-EGFP transgenic lines, in which the reporter genes LacZ and EGFP are expressed under the control of Villin promoter (Qia et al., 2007). Immunohistochemical analysis of stomach section of adult transgenic mice showed that expression of reporter genes is restricted to a few epithelial cells in antrum of the stomach. Most glands did not appear to contain β-gal- or GFP-positive cells, when present; they commonly appeared as single cells in a single gastric unit. After treatment of the transgenic mice with interferon gamma (IFN-γ), Qia et al found that the gastric progenitor cells (β-gal-positive cells) were greatly expanded in number and adjacent gastric gland often contained β-gal-positive cells.

These data revealed that the gastric progenitor cells are normally quiescent, but that IFN-γ induces their mitotic division.

To determine the percentage of GFP-positive cells in stomach during the pre- and postnatal developmental stages, flow cytometric analyses were performed. The GFP-positive cells

constituted around 4.62% of the total cell suspension from whole stomach of E13.5. The percentage of GFP-positive cells increased during the prenatal developmental stages and reached at maximum level of 8.26% in cellular suspension of E18.5. After birth, the percentage of GFP-positive cells was decreased rapidly. This result is also consistent with the previous data about dynamics of stomach stem cell population according to developmental stages.

Establishing of primary cell culture, especially for epithelial stem cells of stomach is very important for characterization of stomach stem cell, studying gastric cancer and Helicobacter induced epithelial alteration. Successful establishment and characterization of stomach epithelial cell line have not yet been reported. Recently, Yang et al (2007) tried to isolate and characterise human epithelial stem cells of stomach. They used special medium (K-NAC) containing 5 ng/ml epidermal growth factor for the cultivation of stomach stem cell suspension. They could isolate several cell clones, which show stem cell morphology.

After the first passage, various morphologically different cell types were observed in the passaged cells besides epithelial-like cells such as round cells, neuron-like and glial-like cells. To prevent the differentiation of the passaged cells, leukemia inhibitory factor (LIF;

10 ng/mL), fibroblast growth factor (FGF; 200 mg/mL), xanthosin (50 mmol/L) and pentagastrin (0.1 mmol/L) were added in the culture medium. However, all these agents failed to prevent the extensive differentiation of these cells (Yang et al., 2007).

We also tried to establish stomach stem cell line from FACS-sorted GFP-positive cells . We used ES-cell medium containing LIF to grow the positive cell. But the most positive cells were not attached to the dish, and also the few number of attached GFP-positive cells lost rapidly their green fluorescence after over night culture. This result suggests that the isolated GFP-positive cells are not able to grow under the used culture conditions.