Prospective Identification of a Multilineage Progenitor in Murine Stomach Epithelium
2007; Elsevier BV; Volume: 133; Issue: 6 Linguagem: Inglês
10.1053/j.gastro.2007.09.031
ISSN1528-0012
AutoresXiaotan T. Qiao, Joshua W. Ziel, Wendy M. McKimpson, Blair Madison, Andrea Todisco, Juanita L. Merchant, Linda C. Samuelson, Deborah L. Gumucio,
Tópico(s)Digestive system and related health
ResumoBackground & Aims: Epithelial stem cells in the stomach are responsible for constant renewal of the epithelium through generation of multiple gastric cell lineages that populate the gastric glands. However, gastric stem or progenitor cells have not been well-characterized because of the lack of specific markers that permit their prospective recognition. We identified an intestinal promoter that is active in a rare subpopulation of gastric epithelial cells and investigated whether these cells possess multilineage potential. Methods: A marked allele of the endogenous mouse villin locus was used to visualize single β-galactosidase–positive cells located in the lower third of antral glands. A 12.4-kb villin promoter/enhancer fragment drives several transgenes (EGFP, β-galactosidase, and Cre recombinase) in these cells in a pattern similar to that of the marked villin allele. Reporter gene activity was used to track these cells during development and to examine cell number in the context of inflammatory challenge while Cre activity allowed lineage tracing in vivo. Results: We show that these rare epithelial cells are normally quiescent, but multiply in response to interferon γ. Lineage tracing studies confirm that these cells give rise to all gastric lineages of the antral glands. In the embryo, these cells are located basally in the stomach epithelium before completion of gastric gland morphogenesis. Conclusions: We have identified a rare subpopulation of gastric progenitors with multilineage potential. The ability to prospectively identify and manipulate such progenitors in situ represents a major step forward in gastric stem cell biology and has potential implications for gastric cancer. Background & Aims: Epithelial stem cells in the stomach are responsible for constant renewal of the epithelium through generation of multiple gastric cell lineages that populate the gastric glands. However, gastric stem or progenitor cells have not been well-characterized because of the lack of specific markers that permit their prospective recognition. We identified an intestinal promoter that is active in a rare subpopulation of gastric epithelial cells and investigated whether these cells possess multilineage potential. Methods: A marked allele of the endogenous mouse villin locus was used to visualize single β-galactosidase–positive cells located in the lower third of antral glands. A 12.4-kb villin promoter/enhancer fragment drives several transgenes (EGFP, β-galactosidase, and Cre recombinase) in these cells in a pattern similar to that of the marked villin allele. Reporter gene activity was used to track these cells during development and to examine cell number in the context of inflammatory challenge while Cre activity allowed lineage tracing in vivo. Results: We show that these rare epithelial cells are normally quiescent, but multiply in response to interferon γ. Lineage tracing studies confirm that these cells give rise to all gastric lineages of the antral glands. In the embryo, these cells are located basally in the stomach epithelium before completion of gastric gland morphogenesis. Conclusions: We have identified a rare subpopulation of gastric progenitors with multilineage potential. The ability to prospectively identify and manipulate such progenitors in situ represents a major step forward in gastric stem cell biology and has potential implications for gastric cancer. The epithelium of the stomach is a continuously renewing tissue that is organized into repeating flask-like tubular extensions called glands. The multiple cell lineages that populate each gland are generated by controlled division of gastric epithelial stem cells located within the gland. Apart from the general importance of the stem cell population in the maintenance and regeneration of the gastric epithelium, it is widely held that the stem or progenitor cell also represents the target for the genetic changes that lead to tumorigenesis (for a review, see Houghton and Wang1Houghton J. Wang T.C. Helicobacter pylori and gastric cancer: a new paradigm for inflammation-associated epithelial cancers.Gastroenterology. 2005; 128: 1567-1578Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar). This hypothesis has not been tested directly, however, because it has not been possible to follow single gastric stem or progenitor cells prospectively. Despite the lack of specific markers to definitively identify gastric stem cells in situ, some of the characteristics of gastric stem cells have been inferred from elegant morphologic and lineage tracing analyses. Labeling studies with thymidine analogues have identified a highly proliferative zone near the isthmus of the gland and electron microscopic studies have revealed immature-looking cells in this region (ie, small cells with prominent nucleoli and lacking secretory granules)2Lee E.R. Leblond C.P. Dynamic histology of the antral epithelium in the mouse stomach: II Ultrastructure and renewal of isthmal cells.Am J Anat. 1985; 172: 205-224Crossref PubMed Scopus (78) Google Scholar; these are the presumed stem cells. The dynamics of stem cell division have been inferred retrospectively by following the patterns of X-chromosome inactivation, by tracking mutant clones after chemical mutagenesis, or by studying the pattern of strain-specific–marker expression in chimeric or transgenic animals.3Lorenz R.G. Gordon J.I. Use of transgenic mice to study regulation of gene expression in the parietal cell lineage of gastric units.J Biol Chem. 1993; 268: 26559-26570Abstract Full Text PDF PubMed Google Scholar, 4Tatematsu M. Fukami H. Yamamoto M. et al.Clonal analysis of glandular stomach carcinogenesis in C3H/HeN⇔BALB/c chimeric mice treated with N-methyl-N-nitrosourea.Cancer Lett. 1994; 83: 37-42Abstract Full Text PDF PubMed Scopus (63) Google Scholar, 5Thompson M. Fleming K.A. Evans D.J. et al.Gastric endocrine cells share a clonal origin with other gut cell lineages.Development. 1990; 110: 477-481PubMed Google Scholar, 6Nomura S. Esumi H. Job C. et al.Lineage and clonal development of gastric glands.Dev Biol. 1998; 204: 124-135Crossref PubMed Scopus (74) Google Scholar, 7Nomura S. Kaminishi M. Sugiyama K. et al.Clonal analysis of isolated single fundic and pyloric gland of stomach using X-linked polymorphism.Biochem Biophys Res Commun. 1996; 226: 385-390Crossref PubMed Scopus (47) Google Scholar, 8Bjerknes M. Cheng H. Multipotential stem cells in adult mouse gastric epithelium.Am J Physiol. 2002; 283: G767-G777Crossref PubMed Scopus (138) Google Scholar, 9Nomura S. Kaminishi M. Sugiyama K. et al.Clonal analysis of isolated intestinal metaplastic glands of stomach using X linked polymorphism.Gut. 1998; 42: 663-668Crossref PubMed Scopus (30) Google Scholar Together, these studies revealed that in the antrum of adult mice and human beings, the majority of glands are functionally monoclonal; that is, all cellular progeny of the glandular epithelium arise from a single stem cell.6Nomura S. Esumi H. Job C. et al.Lineage and clonal development of gastric glands.Dev Biol. 1998; 204: 124-135Crossref PubMed Scopus (74) Google Scholar, 7Nomura S. Kaminishi M. Sugiyama K. et al.Clonal analysis of isolated single fundic and pyloric gland of stomach using X-linked polymorphism.Biochem Biophys Res Commun. 1996; 226: 385-390Crossref PubMed Scopus (47) Google Scholar Mathematic models predict that when murine antral glands initially are established, they contain approximately 3 stem cells.6Nomura S. Esumi H. Job C. et al.Lineage and clonal development of gastric glands.Dev Biol. 1998; 204: 124-135Crossref PubMed Scopus (74) Google Scholar These initially polyclonal glands resolve to a primarily monoclonal state over the first 6 weeks of life,4Tatematsu M. Fukami H. Yamamoto M. et al.Clonal analysis of glandular stomach carcinogenesis in C3H/HeN⇔BALB/c chimeric mice treated with N-methyl-N-nitrosourea.Cancer Lett. 1994; 83: 37-42Abstract Full Text PDF PubMed Scopus (63) Google Scholar, 6Nomura S. Esumi H. Job C. et al.Lineage and clonal development of gastric glands.Dev Biol. 1998; 204: 124-135Crossref PubMed Scopus (74) Google Scholar, 7Nomura S. Kaminishi M. Sugiyama K. et al.Clonal analysis of isolated single fundic and pyloric gland of stomach using X-linked polymorphism.Biochem Biophys Res Commun. 1996; 226: 385-390Crossref PubMed Scopus (47) Google Scholar, 9Nomura S. Kaminishi M. Sugiyama K. et al.Clonal analysis of isolated intestinal metaplastic glands of stomach using X linked polymorphism.Gut. 1998; 42: 663-668Crossref PubMed Scopus (30) Google Scholar although a few residual polyclonal glands (5%–10%) persist in the adult.9Nomura S. Kaminishi M. Sugiyama K. et al.Clonal analysis of isolated intestinal metaplastic glands of stomach using X linked polymorphism.Gut. 1998; 42: 663-668Crossref PubMed Scopus (30) Google Scholar One force that is believed to promote the resolution of a mixed gland to a monoclonal state is gland fission; the gland bifurcates, producing 2 daughter glands, each with half of the stem cell census of the original.10Hattori T. Fjuita S. Fractographic study on the growth and multiplication of the gastric gland of the hamster The gland division cycle.Cell Tissue Res. 1974; 153: 145-149Crossref PubMed Scopus (25) Google Scholar Morphologic evidence of stomach gland fission has been reported in the adult7Nomura S. Kaminishi M. Sugiyama K. et al.Clonal analysis of isolated single fundic and pyloric gland of stomach using X-linked polymorphism.Biochem Biophys Res Commun. 1996; 226: 385-390Crossref PubMed Scopus (47) Google Scholar, 8Bjerknes M. Cheng H. Multipotential stem cells in adult mouse gastric epithelium.Am J Physiol. 2002; 283: G767-G777Crossref PubMed Scopus (138) Google Scholar and in the neonate.6Nomura S. Esumi H. Job C. et al.Lineage and clonal development of gastric glands.Dev Biol. 1998; 204: 124-135Crossref PubMed Scopus (74) Google Scholar The signal that initiates gland fission is unknown, but it has been proposed that intestinal crypts divide in response to a doubling of stem cell number.11Loeffler M. Birke A. Winton D. et al.Somatic mutation, monoclonality and stochastic models of stem cell organization in the intestinal crypt.J Theor Biol. 1993; 160: 471-491Crossref PubMed Scopus (144) Google Scholar, 12Loeffler M. Bratke T. Paulus U. et al.Clonality and life cycles of intestinal crypts explained by a state dependent stochastic model of epithelial stem cell organization.J Theor Biol. 1997; 186: 41-54Crossref PubMed Scopus (76) Google Scholar In fact, recent studies have revealed that in intestines of mice carrying a conditional phosphatase and tensin homolog (PTEN) deletion, stem cell numbers are increased and this appears to directly promote crypt budding as well as crypt fission.13He X.C. Yin T. Grindley J.C. et al.PTEN-deficient intestinal stem cells initiate intestinal polyposis.Nat Genet. 2007; 39: 189-198Crossref PubMed Scopus (375) Google Scholar It is possible that a similar stem cell-dependent counting mechanism might operate in gastric glands. In this report, we identify a novel gastric cell population in situ that has properties similar to those of presumptive gastric stem cell populations described earlier. We call these cells gastric progenitor cells (GPCs). We first identified murine GPCs because they robustly express a marked allele of the Villin gene (Villinβ-gal/+) generated earlier in our laboratory.14Pinson K.I. Dunbar L. Samuelson L. et al.Targeted disruption of the mouse villin gene does not impair the morphogenesis of microvilli.Dev Dyn. 1998; 211: 109-121Crossref PubMed Scopus (82) Google Scholar Villin is an intestinal gene and generally is not expressed in the stomach; GPCs thus appear as rare β-gal–positive cells in an otherwise β-gal–negative adult gastric epithelium. GPCs are found predominantly in the antrum, where they are located at or below the isthmus, the region thought to harbor stem cells. Indeed, we use lineage tracing reagents to show that GPCs can populate entire antral glands with progeny of multiple gastric cell lineages. 5-bromo-2'-deoxyuridine (BrdU) labeling studies suggest that GPCs are normally quiescent, and that they have label-retaining properties; these are thought to be characteristics of stem or progenitor cells. Administration of interferon γ (IFNγ), a potent proinflammatory cytokine, causes GPC amplification. This cytokine-stimulated division of GPC appears to be symmetric (ie, creating more GPCs). Interestingly, the pattern of resultant labeled glands suggests that the increase in GPC number might promote gland fission. Transgenic mice (12.4KVil-EGFP) were generated in the University of Michigan Transgenic Animal Core. These mice, as well as Villinβ-gal/+,14Pinson K.I. Dunbar L. Samuelson L. et al.Targeted disruption of the mouse villin gene does not impair the morphogenesis of microvilli.Dev Dyn. 1998; 211: 109-121Crossref PubMed Scopus (82) Google Scholar 12.4KVil-LacZ,15Madison B.B. Dunbar L. Qiao X.T. et al.Cis elements of the villin gene control expression in restricted domains of the vertical (crypt) and horizontal (duodenum, cecum) axes of the intestine.J Biol Chem. 2002; 277: 33275-33283Crossref PubMed Scopus (601) Google Scholar 12.4KVil-Cre,15Madison B.B. Dunbar L. Qiao X.T. et al.Cis elements of the villin gene control expression in restricted domains of the vertical (crypt) and horizontal (duodenum, cecum) axes of the intestine.J Biol Chem. 2002; 277: 33275-33283Crossref PubMed Scopus (601) Google Scholar Ctox7 transgenic mice,16Lopez-Diaz L. Hinkle K.L. Jain R.N. et al.Parietal cell hyperstimulation and autoimmune gastritis in cholera toxin transgenic mice.Am J Physiol. 2006; 290: G970-G979Crossref PubMed Scopus (45) Google Scholar Cdx2 transgenic mice,17Silberg D.G. Sullivan J. Kang E. et al.Cdx2 ectopic expression induces gastric intestinal metaplasia in transgenic mice.Gastroenterology. 2002; 122: 689-696Abstract Full Text Full Text PDF PubMed Scopus (414) Google Scholar and ROSA26R mice18Soriano P. Generalized lacZ expression with the ROSA26 Cre reporter strain.Nat Genet. 1999; 21: 70-71Crossref PubMed Scopus (4215) Google Scholar were maintained in our University Committee on the Use and Care of Animals-approved facility. All procedures were performed in accord with previously approved guidelines. All lines were maintained continuously on a C57BL/6J background. Surgically implanted Alzet microosmotic pumps (Durect Corporation, Cupertio, CA) were loaded with 250 U/kg INFγ (R&D Systems, Minneapolis, MN) in phosphate-buffered saline (PBS) with 1% bovine serum albumin as previously described.19Kang W. Rathinavelu S. Samuelson L.C. et al.Interferon gamma induction of gastric mucous neck cell hypertrophy.Lab Invest. 2005; 85: 702-715Crossref PubMed Scopus (76) Google Scholar Control mice received pumps containing PBS with 1% bovine serum albumin. Animals were killed at 2, 8, or 12 weeks after pump implantation. In some experiments, mice were injected intraperitoneally with IFNγ (17 IU/kg in PBS with 1% bovine serum albumin) on a daily basis for 7–14 days before death. Stomachs were removed, opened along the greater curvature, washed with PBS, and incubated in 30 mmol/L ethylenediaminetetraacetic acid in Hank's balanced salt solution for 15 minutes at 37°C. The stomach then was pinned to a wax surface and rinsed with Hank's balanced salt solution. To loosen glands, excess Hank's balanced salt solution was applied and a plastic pipette was used to gently aspirate and expel the liquid several times until glands were freed from the underlying stroma. Large mats of connected glands were separated further by trituration. Glands were fixed in 4% paraformaldehyde for 5 minutes before staining with X-gal. Methods for tissue preparation, whole mount, or section X-gal staining were as previously described.20Madison B.B. Braunstein K. Kuizon E. et al.Epithelial hedgehog signals pattern the intestinal crypt-villus axis.Development. 2005; 132: 279-289Crossref PubMed Scopus (307) Google Scholar, 21Braunstein E.M. Qiao X.T. Madison B. et al.Villin: a marker for development of the epithelial pyloric border.Dev Dyn. 2002; 224: 90-102Crossref PubMed Scopus (44) Google Scholar Frozen or paraffin sections were used for antibody and/or lectin staining. Lectins used were as follows: Griffonia simplicifolia II (GSII) and Ulex europaeus agglutinin I (UEAI) (both 1:200; Vector Laboratories, Burlingame, CA). Antibodies used for cell identification were rabbit anti-H+, K+ adenosine triphosphatase (ATPase) α subunit (1:500; Medical and Biological Laboratories, Nagoya, Japan), rabbit anti–β-galactosidase (kind gift of J. Douglas Engel, Department of Cell and Developmental Biology, University of Michigan; used at 1:500); mouse anti-BrdU (1:400; (Developmental Studies Hybridoma Bank, Iowa City, IA)); goat antiserotonin (1:250, diluted in PBS, 0.1% triton; ImmunoStar, Hudson, WI); rabbit anti-GFP (1:500; Invitrogen, Carlsbad, CA); and chicken anti-GFP (1:500; ABCam, Cambridge, MA). Images were obtained on a Nikon Eclipse E800 microscope (Nikon, Melville, NY) using a Spot CD camera. Confocal microscopy was performed using an Olympus FV-500 confocal microscope (Olympus, Center Valley, PA). For pulse-labeling studies, BrdU (Sigma, St. Louis, MO) was administered intraperitoneally (100 μg/g/body weight) in 0.1 mol/L PBS with 1% bovine serum album (Sigma) 1 hour before death. For long-term labeling, mice were given one injection of BrdU as described previously, and then were kept on oral administration of BrdU in the drinking water (2.2 mg/mL as previously described22Santoso A. Kaiser A. Winter Y. Individually dosed oral drug administration to socially-living transponder-tagged mice by a water dispenser under RFID control.J Neurosci Methods. 2006; 153: 208-213Crossref PubMed Scopus (19) Google Scholar) for 7 days before death. Some animals were provided with regular tap water for another 7 days (chase period). Stomachs were removed, fixed in 4% paraformaldehyde (PFA) on ice for 10 minutes, washed in PBS (pH 7.4), snap-frozen in O.C.T. (Electron Microscopy Sciences, Hatfield, PA), and stored at −80°C before use. Air-dried sections were washed in PBS, acid denatured in 2 N HCL for 15 minutes, neutralized, and further washed in PBS before immunostaining. Our laboratory previously generated mice that carry a marked villin allele (Villinβ-gal/+).14Pinson K.I. Dunbar L. Samuelson L. et al.Targeted disruption of the mouse villin gene does not impair the morphogenesis of microvilli.Dev Dyn. 1998; 211: 109-121Crossref PubMed Scopus (82) Google Scholar In adult mice, villin (and β-gal in the case of the marked allele) is highly expressed in intestinal cells, whereas the gastric epithelium is largely devoid of villin. However, careful analysis of Villinβ-gal/+ mice revealed rare β-gal–positive cells in the antrum of the stomach (compare Figure 1A and B). The cells were located in the bottom third of the antral glands and were oval or triangular in shape with a large pale nucleus (Figure 1B inset, and C). Most glands did not appear to contain β-gal–positive cells; when present, they commonly appeared as a single cell per sectioned gland, or rarely as 2 β-gal cells in a single gland (Figure 1D). To document the number and distribution of these cells, stomachs of 3-month-old Villinβ-gal/+ mice were opened along the greater curvature and each entire stomach was sectioned serially across the length of the stomach. The number of β-gal–positive cells in every section was counted. For 3 animals, 192, 414, and 292 β-gal–positive cells were found, the majority of which were located on the lesser curvature of the stomach, just lateral to the ventral midline. Within individual glands (Figure 1E and F), the majority of β-gal–positive cells were located in the antrum, at or below the isthmus of the glands. Because the paucity of these cells in the corpus made them difficult to study in sufficient numbers, the remainder of the analysis here is concentrated on the β-gal–positive cells found in the antrum. The lesser curvature of the antrum is a common site for gastric tumor formation in human beings23Odze R.D. Unraveling the mystery of the gastroesophageal junction: a pathologist's perspective.Am J Gastroenterol. 2005; 100: 1853-1867Crossref PubMed Scopus (111) Google Scholar as well as in several mouse models of gastric cancer.24Zavros Y. Eaton K.A. Kang W. et al.Chronic gastritis in the hypochlorhydric gastrin-deficient mouse progresses to adenocarcinoma.Oncogene. 2005; 24: 2354-2366Crossref PubMed Scopus (123) Google Scholar, 25Judd L.M. Alderman B.M. Howlett M. et al.Gastric cancer development in mice lacking the SHP2 binding site on the IL-6 family co-receptor gp130.Gastroenterology. 2004; 126: 196-207Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 26Cai X. Carlson J. Stoicov C. et al.Helicobacter felis eradication restores normal architecture and inhibits gastric cancer progression in C57BL/6 mice.Gastroenterology. 2005; 128: 1937-1952Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar Commonly, the pathologic cascade for gastric cancer27Correa P. Helicobacter pylori and gastric cancer: state of the art.Cancer Epidemiol Biomarkers Prev. 1996; 5: 477-481PubMed Google Scholar begins with chronic inflammation and progresses through parietal cell atrophy, metaplasia (intestinal metaplasia or mucous metaplasia called spasmolytic polypeptide-expressing metaplasia10Hattori T. Fjuita S. Fractographic study on the growth and multiplication of the gastric gland of the hamster The gland division cycle.Cell Tissue Res. 1974; 153: 145-149Crossref PubMed Scopus (25) Google Scholar, 28Schmidt P.H. Lee J.R. Joshi V. et al.Identification of a metaplastic cell lineage associated with human gastric adenocarcinoma.Lab Invest. 1999; 79: 639-646PubMed Google Scholar), to dysplasia, and, finally, tumors. To determine whether the number or location of β-gal–positive cells is altered by the metaplastic progression, we examined these cells in the context of 3 mouse models characterized by metaplasia: (1) Cdx2 transgenic mice,17Silberg D.G. Sullivan J. Kang E. et al.Cdx2 ectopic expression induces gastric intestinal metaplasia in transgenic mice.Gastroenterology. 2002; 122: 689-696Abstract Full Text Full Text PDF PubMed Scopus (414) Google Scholar (2) Ctox-7 transgenic mice,16Lopez-Diaz L. Hinkle K.L. Jain R.N. et al.Parietal cell hyperstimulation and autoimmune gastritis in cholera toxin transgenic mice.Am J Physiol. 2006; 290: G970-G979Crossref PubMed Scopus (45) Google Scholar and (3) IFNγ-treated mice.19Kang W. Rathinavelu S. Samuelson L.C. et al.Interferon gamma induction of gastric mucous neck cell hypertrophy.Lab Invest. 2005; 85: 702-715Crossref PubMed Scopus (76) Google Scholar Cdx2, a caudal-related homeobox transcription factor, is a master regulator of intestinal differentiation; transgenic mice ectopically expressing Cdx2 in gastric epithelium show widespread intestinal metaplasia of the stomach.17Silberg D.G. Sullivan J. Kang E. et al.Cdx2 ectopic expression induces gastric intestinal metaplasia in transgenic mice.Gastroenterology. 2002; 122: 689-696Abstract Full Text Full Text PDF PubMed Scopus (414) Google Scholar Intestinal metaplasia was documented in the antrum of 3-month-old Cdx2 transgenic mice by alkaline phosphatase staining in cells of the upper third of antral glands (Figure 2A). X-gal staining of adjacent sections showed that the villin β-gal marker was expressed in some surface cells (Figure 2B), likely reflecting activation of the villin promoter in the metaplastic cells. However, the number, location, and distribution of β-gal–positive cells located deep in the antral glands were similar in Cdx2 transgenic mice and their wild-type littermates; deep-gland β-gal–positive cells were not found in every gland with metaplastic changes. Thus, it seems unlikely that the deep gland β-gal cells are the source of the intestinal metaplasia in this model. Ctox-7 mice express the cholera toxin A1 subunit under control of the parietal cell-specific H+,K+-ATPase β-subunit promoter. These mice suffer parietal and chief cell loss and develop inflammatory infiltrates with high IFNγ expression and extensive mucous cell metaplasia, located primarily in the corpus.16Lopez-Diaz L. Hinkle K.L. Jain R.N. et al.Parietal cell hyperstimulation and autoimmune gastritis in cholera toxin transgenic mice.Am J Physiol. 2006; 290: G970-G979Crossref PubMed Scopus (45) Google Scholar We recently identified antral tumors in some of these mice (L.C.S., X.T.Q., unpublished data). Ctox-7 mice also show intestinal metaplasia of the corpus (data not shown) as well as focal regions of the antrum (Figure 2C). In contrast to the finding with Cdx2 mice, the population of antral β-gal–positive cells was greatly expanded in the Ctox-7 model (compare Figures 2D and E with Figure 1B). Quantitation of cells in the entire stomach of these mice revealed 6797 ± 1523 cells (mean ± SD; n = 3), a dramatic increase over the number seen in the wild-type stomach (mean = 336, see earlier). The pattern of labeled cells within the glands also was altered: in wild-type mice the glands containing labeled cells were spaced widely, whereas in Ctox-7 mice the adjacent glands often contained labeled cells (Figure 2D). However, amplified β-gal–positive cells in Ctox-7/Villinβ-gal/+ mice remained concentrated on the lesser curvature of the antrum and did not appear to follow the focal distribution of intestinal metaplasia in the antrum. Finally, surgical implantation of osmotic minipumps that deliver IFNγ into the peritoneum for 2 weeks produces gastritis, along with an expansion of spasmolytic polypeptide-expressing metaplasia–like cells in the corpus of the stomach.19Kang W. Rathinavelu S. Samuelson L.C. et al.Interferon gamma induction of gastric mucous neck cell hypertrophy.Lab Invest. 2005; 85: 702-715Crossref PubMed Scopus (76) Google Scholar As in the Ctox-7 model (which also shows high IFNγ levels), β-gal–positive cells were expanded markedly along the lesser curvature of the antrum in IFNγ-treated animals (Figure 2F). In support of this, more examples of glands with 2 β-gal–positive cells were seen in both the IFNγ and Ctox-7 models (eg, Figure 2D, arrow) as compared with wild-type mice. Quantitation of β-gal–positive cells by serial sectioning in 3 IFNγ-infused mice and 3 PBS-infused littermates revealed 1431 ± 477 cells and 175 ± 33 cells, respectively (mean ± SD). Although we have not ruled out the possibility that metaplastic cells can arise from β-gal–positive cells, this seems unlikely given the different distributions of these 2 cell types in the 3 models. However, it appears that IFNγ itself, or some aspect of the proinflammatory cascade downstream of this cytokine, is a potent signal for amplification of the β-gal–marked cells. Because inflammation is connected closely to the evolution of gastric cancer, this finding potentially could signify a link between the concentration of β-gal–positive cells on the lesser curvature and the frequency of tumors in this location. Expansion of β-gal–positive cells could result either from an IFNγ-induced mitotic stimulus, or could reflect stress-induced activation of the Villinβ-gal/+ allele in postmitotic cells. To clarify this, we performed BrdU-labeling studies in animals treated with IFNγ. These experiments (supplemental Figure 1A–I; see supplementary material online at www.gastrojournal.org) revealed that β-gal–positive cells are normally quiescent, but that IFNγ induces their mitotic division. Furthermore, β-gal–positive cells are among the few label-retaining cells detected in antral gland epithelium (supplementary Figure 1J–M; see supplementary material online at www.gastrojournal.org). To better assess the number and location of β-gal–positive cells, we isolated whole glands and examined them under a dissecting microscope. Even after IFNγ treatment, many glands lacked β-gal staining cells. In glands with marked cells, most contained 1 (Figure 3E and I) and a few contained 2 (Figure 3F–H, and J) β-gal–positive cells. In 1 case (Figure 3F), 2 closely adjoined cells were seen on the long axis of the gland, the apparent products of a recent cell division. Marked cells were located at variable positions in the lower portion of the gland, between the isthmus and the gland tip (Figure 3A–E). In several instances, we observed 2 β-gal–positive cells situated on opposite sides of the base of the gland (Figure 3G, H, and J; also see Figure 1D). Because gland fission has been shown previously to initiate from the base of the antral glands,6Nomura S. Esumi H. Job C. et al.Lineage and clonal development of gastric glands.Dev Biol. 1998; 204: 124-135Crossref PubMed Scopus (74) Google Scholar a gland fission event would cleanly partition these cells into 2 different glands. In the intestine, crypt fission is believed to be a response to increased stem cell number11Loeffler M. Birke A. Winton D. et al.Somatic mutation, monoclonality and stochastic models of stem cell organization in the intestinal crypt.J Theor Biol. 1993; 160: 471-491Crossref PubMed Scopus (144) Google Scholar, 12Loeffler M. Bratke T. Paulus U. et al.Clonality and life cycles of intestinal crypts explained by a state dependent stochastic model of epithelial stem cell organization.J Theor Biol. 1997; 186: 41-54Crossref PubMed Scopus (76) Google Scholar and the rate of fission of colonic crypts is greatly increased in inflammatory states such as Crohn's disease and ulcerative colitis.29Cheng H. Bjerknes M. Amar J. et al.Crypt production in normal and diseased human colonic epithelium.Anat Rec. 1986; 216: 44-48Crossref PubMed Scopus (77) Google Scholar These observations are intriguing in light of the ability of IFNγ to cause amplification of these β-gal–positive cells. A ra
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