**2.1. Discovery of gastric stem cells and their markers**

recurrence, and metastasis. Herein, we first review the major markers of stem/progenitor cells in the stomach, then we describe the cells at the origin of gastric tumors, and finally, we focus

In the stomach, the gastric epithelium is a physiologically self-renewing tissue with a cycle of 2–7 days. Anatomically, the stomach is divided into three main parts: the cardiac region (in humans) or the forestomach (in mice), the main body (corpus), and the distal part (antrum/ pylorus). The mucosa of the stomach is composed of a glandular epithelium with millions of gastric units. Each gland is considered to be monoclonal [1] and is subdivided into the foveolus, isthmus, neck, and bottom regions (**Figure 1**). In the gastric corpus, the glands are long and, from the bottom to the top of the gland, contain zymogenic/chief cells implicated in digestion, parietal cells that are essential for acid production, enterochromaffin-like cells that control acid production, mucous neck cells, and superficial pit cells. In the antrum, the glands are shorter and are composed mainly of mucus-producing cells and enteroendocrine hormone-secreting cells that regulate acid and digestive enzyme production in the corpus. In both regions, some discrete gastric stem cells exist and are instrumental in stomach epithe-

**Corpus gland**

Base **Troy+**

**Villin+**

CD44+

**Lgr5+** *Sox2* +

**Figure 1.** Architecture of the gastric glands and localization of stem/progenitor cells in the main parts of the stomach. (a) Fundic/corpus gastric gland. (b) Antral/pyloric gastric gland. Stem/progenitor markers identified by lineage tracing

*Sox2+*

*Stem/progenitor cells*

*CD44+* **Mist1+** *Stem/progenitor*

*Reservoir of quiescent*

*cells*

*stem cells*

*TFF2+*

Isthmus

Neck

Surface

**a**

on the characterization of the GCSC subpopulation.

64 Gastric Cancer

**2. Existence of stem cells in the stomach**

lium renewal under pathophysiological conditions.

Fundus/ Corpus

Antrum/ Pylorus

**gland**

**b**

are indicated in bold.

**Antral/pyloric**

Using radiolabeling experiments and analyses of the cells by electron microscopy, Leblond et al. first identified a group of small undifferentiated and granule-free cells with the highest labeling index as the putative stem/progenitor cells. These cells are localized in the isthmus region from where they migrate toward both the pit and the bottom [3, 4]. However, the first evidence of the existence of multipotent stem cells in adult mouse gastric glands was found later using chemical mutagenesis of single cells and long-term gastric epithelial cell analyses where many clones spanned entire glands containing all specialized gastric cell lineages [5]. The use of inducible Cre recombinase activity to indelibly label putative stem/progenitor cells and their progeny in the stomach has been widely practiced and is considered as the gold standard method for lineage tracing studies. The first marker of gastric stem/progenitor cells revealed by lineage tracing in the gastric mucosa was **Villin**. *Villin-lacZ* transgenic mice revealed a rare population of quiescent β-galactosidase-positive cells located at the bottom of antropyloric glands or at the isthmus in the corpus [6]. These quiescent Villin<sup>+</sup> cells can be activated after stimulation by interferon-γ and moved from the isthmus toward the base of the gland to generate all of the specialized cells of the gastric glands. Villin<sup>+</sup> cells may act as a stem cell reservoir with a high proliferative potential to regenerate the gastric mucosa after injury. The presence of such a cell population that highly responds to inflammation is very interesting, especially in the context of gastric cancer which is initiated by a chronic inflammation of the gastric mucosa.

Leucine-rich G protein-coupled receptor 5 (**Lgr5**), a well-recognized stem cell maker in the intestine, is expressed at the bottom of prospective corpus and pyloric glands in the stomach of neonates, whereas its expression in adults is predominantly restricted to the bottom of pyloric glands in mice and in humans [7]. Lineage tracing experiments revealed that Lgr5<sup>+</sup> cells are multipotent stem cells responsible for the long-term renewal of the gastric epithelium. In vitro, single Lgr5<sup>+</sup> cells generated long-lived organoids resembling the pyloric epithelium in three-dimensional culture. Lgr5<sup>+</sup> cells divide symmetrically to generate clonal gland units via neutral competition and lateral expansion of stem cell clones via gland fission under non-damaging conditions [8].

These two markers identified stem cells in the gastric antrum/pyloric region, where most of distal gastric carcinoma arises in humans. In the corpus, some studies suggested that **Sox2**<sup>+</sup> cells may represent long-lived stem cells scattered throughout the isthmus and in the lower part of the gastric unit [9]. Trefoil factor family 2 (**Tff2**<sup>+</sup> ) cells were also described as shortlived progenitors in the isthmus region of the corpus [10]. More recently, lineage tracing experiments have shown that differentiated mature chief cells expressing the **Troy** marker at the base of the corpus gastric glands can generate entirely labeled gastric units over a period of several months in vivo and long-lived organoids in vitro [11]. This phenomenon is accelerated upon depletion of the proliferating isthmus compartment mediated by 5-fluorouracile treatment, suggesting that the gastric corpus also seems to contain two stem cell populations: (1) an actively dividing population located in the isthmus region and (2) a smaller reserve population of *Troy+* stem-like chief cells located at the base of the gland [11]. This property of a differentiated cell to reenter the cell cycle and to act as a multipotent stem cell highlights the plasticity of gastric epithelial cells. Surprisingly, Stange et al. detected Lgr5<sup>+</sup> cells at the base of the corpus glands using another Lgr5 reporter construction in transgenic mice, and transcriptomic analyses demonstrated that Troy<sup>+</sup> cells expresses several Wnt target genes including Lgr5 and CD44 [11].

Likewise, **Mist1** is a marker of stem-like quiescent chief cells located in the lower third of the glands and in rare single cells of the isthmus in the gastric corpus [12, 13]. The vast majority of Mist1<sup>+</sup> chief cells at the base of the glands are Lgr5<sup>+</sup> , whereas Mist1<sup>+</sup> cells in the isthmus are Lgr5− , and only 1.1% of them are proliferative. Ablation of Lgr5<sup>+</sup> /Mist1<sup>+</sup> chief cells by expression of the diphtheria toxin in *Lgr5*-DTR-GFP transgenic mice results in an increase of Mist1<sup>+</sup> cells in the isthmus which reconstitute the entire glands, suggesting that Mist1<sup>+</sup> isthmus cells are multipotent stem cells [13]. Finally, Mist1<sup>+</sup> isthmus cells can form organoids in an Lgr5 independent manner in the corpus.

In addition, the Runx1 enhancer element, **eR1**, is expressed in the isthmus and marks a small number of terminally differentiated chief cells at the base in the stomach corpus as well as near the bottom of the pyloric gland. eR1<sup>+</sup> cells generated entirely labeled gastric units after a year and formed organoids in vitro, suggesting that they are composed of gastric stem cells [14]. Nevertheless, it appears that some Runx1-expressing cells are stem cells, whereas others are differentiated cells, such as pit cells. Moreover, 80% of eR1<sup>+</sup> cells expressed Ki67, whereas only 1.1% of Mist1<sup>+</sup> cells in the isthmus expressed it, suggesting that Mist1<sup>+</sup> cells are quiescent cells and that eR1<sup>+</sup> cells are rapidly dividing cells [13, 14].

Additional markers have been proposed for gastric stem cells (e.g., DCKL1/DCAMKL1, CD133/PROM1, and CD44), but the multipotency of these cells has not yet been analyzed by lineage tracing [15, 16]. Khurana et al. found that **CD44** (cluster of differentiation 44) is mainly expressed at the base of antral/pyloric glands, in a region overlapping Lgr5, and in the isthmus region of the corpus glands [17, 18]. When parietal cell loss and atrophy were induced chemically or by *Helicobacter* infection, the CD44<sup>+</sup> cells expanded from the isthmus and replenished the base of the gastric units (**Figure 2**). CD44 expression is enriched in the Mist1<sup>+</sup> isthmus stem cell population in the corpus, suggesting again that they could represent stem/progenitor cells.

**Figure 2.** CD44 expression in *H. pylori*-induced metaplasia and dysplasia. Representative pictures of CD44 detection by immunohistochemistry in mouse stomachs: (a) normal gastric mucosa of a noninfected mouse; (b) metaplasia; and (c) intraepithelial dysplasia in *H. pylori SS1-*infected stomachs. Scale bars, 50 μm.
