**2.2. Factors sustaining gastric stem cell self-renewal and multipotency**

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

lived 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

a differentiated cell to reenter the cell cycle and to act as a multipotent stem cell highlights the

the corpus glands using another Lgr5 reporter construction in transgenic mice, and transcrip-

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

sion of the diphtheria toxin in *Lgr5*-DTR-GFP transgenic mice results in an increase of Mist1<sup>+</sup>

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

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

cells in the isthmus expressed it, suggesting that Mist1<sup>+</sup>

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

and replenished the base of the gastric units (**Figure 2**). CD44 expression is enriched in the

isthmus stem cell population in the corpus, suggesting again that they could represent

plasticity of gastric epithelial cells. Surprisingly, Stange et al. detected Lgr5<sup>+</sup>

chief cells at the base of the glands are Lgr5<sup>+</sup>

are differentiated cells, such as pit cells. Moreover, 80% of eR1<sup>+</sup>

induced chemically or by *Helicobacter* infection, the CD44<sup>+</sup>

cells are rapidly dividing cells [13, 14].

, and only 1.1% of them are proliferative. Ablation of Lgr5<sup>+</sup>

cells in the isthmus which reconstitute the entire glands, suggesting that Mist1<sup>+</sup>

stem-like chief cells located at the base of the gland [11]. This property of

cells expresses several Wnt target genes including

/Mist1<sup>+</sup>

isthmus cells can form organoids in an Lgr5-

cells generated entirely labeled gastric units after a

, whereas Mist1<sup>+</sup>

) cells were also described as short-

cells at the base of

cells in the isthmus are

cells expressed Ki67, whereas

cells expanded from the isthmus

cells are quiescent

chief cells by expres-

isthmus cells

part of the gastric unit [9]. Trefoil factor family 2 (**Tff2**<sup>+</sup>

population of *Troy+*

66 Gastric Cancer

Lgr5 and CD44 [11].

only 1.1% of Mist1<sup>+</sup>

cells and that eR1<sup>+</sup>

stem/progenitor cells.

Mist1<sup>+</sup>

of Mist1<sup>+</sup>

Lgr5−

tomic analyses demonstrated that Troy<sup>+</sup>

are multipotent stem cells [13]. Finally, Mist1<sup>+</sup>

near the bottom of the pyloric gland. eR1<sup>+</sup>

independent manner in the corpus.

Until very recently, suitable models to study gastric stem cells in vitro were lacking. Cell lines from cell banks are all derived from carcinomas, and primary culture of gastric epithelial cells from biopsies is not successful under conventional adherent culture conditions. Culture of antrum and fundus cells has been rendered possible very recently by the development of mouse and human protocols allowing the development of organoids, named gastroids, under three-dimensional culture conditions, in media containing epithelial growth factor (EGF) and Noggin, with either Wnt3A and R-spondin, a molecule binding Lgr4/5 and potentiating Wnt/β-catenin activity [19], or supplemented with the Notch ligand Jagged-1 [7, 13]. In vitro studies of organoid formation by gastric stem cells or gastric glands have allowed insight in the necessary growth factors and signaling molecules of the niche implicated in stem cell properties and gland formation and can offer new therapeutic applications in patient that suffer malignant diseases, for example, for ulcer treatment. Engevik et al. have shown that gastric stem cells/organoid isolated from young mice can be transplanted into sites of acetic acid-induced ulcer within the stomachs of older mice and that this results in accelerated repair injury [20].

Wnt5a, a noncanonical Wnt ligand, is highly expressed by Cxcr4<sup>+</sup> cells in the isthmus part of the corpus. Histological analyses show that Wnt5A is secreted by Cxcr4<sup>+</sup> resident hematopoietic cells recruited to the isthmus and stimulated by Cxcl12 endothelial cell production. The efficiency of organoid formation is enhanced by Wnt5a or coculture with Cxcr4<sup>+</sup> intraepithelial gastric innate lymphoid cells [13], suggesting that cells in the niche regulate stem/progenitor proliferation.

The enteric nervous system also has the ability to regulate gastric homeostasis via direct innervation of the glands. In three independent mouse models of gastric cancer, Zhao et al. elegantly demonstrated that surgical or pharmacological denervation suppresses gastric tumorigenesis, even if performed at an early preneoplastic step [21]. Further analyses revealed that cholinergic nerves surround the base of glands and modulate epithelial stem cells through activation of the Wnt signaling pathway via the muscarinic acetylcholine receptor 3 (M<sup>3</sup> R) expressed by Lgr5<sup>+</sup> cells. In stomach organoid models, coculture with neurons or treatment with pilocarpine, a cholinomimetic drug, increased organoid formation and the expression of *Lgr5* and *Cd44* stem cell markers, whereas the effects were reversed by botox treatment [21]. Another publication reported that Dclk1<sup>+</sup> tuft cells and nerves, the main sources of acetylcholine in the gastric mucosa, induced nerve growth factor (NGF) secretion from epithelial cells that expand enteric nerves and promote carcinogenesis [22]. Remarkably, *Tff2-*Cre;*R29-*NGF mice developed metaplasia and dysplasia by 8 months of age with CD44<sup>+</sup> dysplastic cell expansion and intramucosal adenocarcinomas by 18 months. Ablation of Dclk1<sup>+</sup> cells in this context led to the inhibition of epithelial proliferation and tumorigenesis in a M<sup>3</sup> R-dependent manner [22].

The Notch signaling pathway is also inhibited in vagotomized mice [21]. The Notch inhibitor dibenzazepine (DBZ) reduced the proliferation in the isthmus region, decreased the Mist1 lineage tracing, and blocked the growth of corpus organoids in vitro, suggesting that Notch activity is important for corpus gastric stem cell maintenance and activity [13].
