Helicobacter pylori Activates and Expands Lgr5+ Stem Cells Through Direct Colonization of the Gastric Glands

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Helicobacter pylori Activates and Expands Lgr5D Stem Cells Through Direct Colonization of the Gastric Glands Michael Sigal,1,2,3 Michael E. Rothenberg,4,5 Catriona Y. Logan,5,6,7 Josephine Y. Lee,2 Ryan W. Honaker,1,2 Rachel L. Cooper,1,2 Ben Passarelli,5 Margarita Camorlinga,8 Donna M. Bouley,9 Guillermo Alvarez,8,10 Roeland Nusse,5,6,7 Javier Torres,8 and Manuel R. Amieva1,2 1

Department of Pediatrics, Division of Infectious Diseases, 2Department of Microbiology and Immunology, 4Department of Medicine, Division of Gastroenterology, 5Institute for Stem Cell Biology, 6Department of Developmental Biology, and 9 Department of Comparative Medicine, Stanford University, Stanford, California; 3Department of Gastroenterology and Hepatology, Charité University Medicine, Berlin, Germany; 7Howard Hughes Medical Institute, Chevy Chase, Maryland; 8 Infectious Disease Research Unit, UMAE Pediatrics, IMSS, Mexico City, Mexico; and 10Endobariatric Surgery, Piedras Negras, Coahuila, Mexico BACKGROUND & AIMS: Helicobacter pylori infection is the main risk factor for gastric cancer. We characterized the interactions of H pylori with gastric epithelial progenitor and stem cells in humans and mice and investigated how these interactions contribute to H pylori–induced pathology. METHODS: We used quantitative confocal microscopy and 3-dimensional reconstruction of entire gastric glands to determine the localizations of H pylori in stomach tissues from humans and infected mice. Using lineage tracing to mark cells derived from leucine-rich repeat-containing G-protein coupled receptor 5–positive (Lgr5þ) stem cells (Lgr5-eGFP-IRESCreERT2/Rosa26-TdTomato mice) and in situ hybridization, we analyzed gastric stem cell responses to infection. Isogenic H pylori mutants were used to determine the role of specific virulence factors in stem cell activation and pathology. RESULTS: H pylori grow as distinct bacterial microcolonies deep in the stomach glands and interact directly with gastric progenitor and stem cells in tissues from mice and humans. These gland-associated bacteria activate stem cells, increasing the number of stem cells, accelerating Lgr5þ stem cell proliferation, and up-regulating expression of stem cell–related genes. Mutant bacteria with defects in chemotaxis that are able to colonize the stomach surface but not the antral glands in mice do not activate stem cells. In addition, bacteria that are unable to inject the contact-dependent virulence factor CagA into the epithelium colonized stomach glands in mice, but did not activate stem cells or produce hyperplasia to the same extent as wild-type H pylori. CONCLUSIONS: H pylori colonize and manipulate the progenitor and stem cell compartments, which alters turnover kinetics and glandular hyperplasia. Bacterial ability to alter the stem cells has important implications for gastrointestinal stem cell biology and H pylori–induced gastric pathology.

gastric cancer, which is the third most lethal cancer worldwide.2 H pylori cannot survive in the acidic lumen of the stomach. Instead, it has evolved multiple mechanisms to persist as free-swimming bacteria in the narrow protective mucus layer that overlays the stomach surface.3 H pylori also use multiple molecular adhesins to attach to the surface of epithelial cells.4,5 After attachment, the bacteria can modify the cell surface to utilize it as a site for replication, where they grow as cell-associated microcolonies.6,7 The more virulent strains of H pylori use a macromolecular syringe, a type IV secretion system (TFSS), to inject the effector molecule CagA directly into host epithelial cells.8 CagA has multiple effects on epithelial cells, including prevention of the assembly of apical junctions, alteration of apicobasal polarity9,10 and activation of multiple signaling molecules downstream of receptor tyrosine kinase growth factors.11,12 Colonization of the epithelial cell surface coupled with CagA delivery is an important persistence strategy for the bacteria,7 but it is also associated with epithelial hyperplasia and malignant transformation, as has been shown in animal models13,14 and in epidemiologic studies of human populations.15 An unsolved question is how H pylori cause these effects on the gastric epithelium in vivo, because most studies describe H pylori adhered to the surface mucus cells, which are terminally differentiated and not long-lived. We recently reported an additional population of H pylori that penetrate and grow as cellassociated microcolonies deep in the gastric glands.16 We asked whether interaction with long-lived cells deeper in the glands has consequences for the host. Recently, leucine-rich repeat-containing G-protein coupled receptor 5 (Lgr5) has been described as a marker of

Keywords: Progenitor Cells; Infection; CagA; Proliferation.

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elicobacter pylori are gram-negative, spiral-shaped bacteria that colonize the stomachs of about 50% of the world population. Although most individuals remain asymptomatic, chronic infection with H pylori is the main risk factor for development of peptic ulcers1 and

Abbreviations used in this paper: 3D, 3-dimensional; GFP, green fluorescent protein; Lgr5, leucine-rich repeat-containing G-protein coupled receptor 5; mRNA, messenger RNA; TFSS, type IV secretion system; WT, wild type. © 2015 by the AGA Institute 0016-5085/$36.00 http://dx.doi.org/10.1053/j.gastro.2015.02.049

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antral long-lived stem cells.17 Lgr5þ stem cells in the base of antral glands drive the repopulation of the glands by giving rise to highly proliferative progenitor cells located in the midglandular compartment that rapidly divide and differentiate into all epithelial lineages.17 Here we provide a detailed characterization of the glandassociated H pylori population in human stomachs and in an experimental mouse model of H pylori infection. In the mouse, we have the ability to mark the gastric stem cells in the antrum as a tool for following their behavior17 in response to H pylori infection. We document that H pylori directly colonize the surface of proliferating progenitor cells as well as Lgr5þ stem cells. We examine the effects of this gland-associated bacterial population on Lgr5– stem cell numbers and proliferation, and study when and how glandassociated H pylori affect the kinetics of stem cells, gland turnover, and the hyperplastic responses that accompany H pylori infection.

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Experimental Procedures To visualize and quantify H pylori in the human and murine mucosa we used confocal microscopy and 3-dimensional (3D) reconstruction of entire gastric glands. To study the effects of H pylori infection on gastric stem cells, we infected Lgr5-eGFP-IRES-CreERT2/Rosa26-TdTomato heterozygous mice18 with a single oral dose of 108 H pylori strain PMSS1 for various times, and analyzed infected tissues as described. Details on all procedures can be found in the Supplementary Material.

Results H pylori Colonize the Progenitor and Stem Cell Compartment in the Murine Stomach

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We used confocal microscopy and 3D reconstruction to characterize the localization of gland-associated H pylori within the murine stomach. At 2 weeks of infection, all mice had free-swimming H pylori in the surface mucus (Figure 1A, arrows), as well as in distinct cell-associated microcolonies deep in the gastric glands (Figure 1A–F). We found the largest gland-associated H pylori microcolonies concentrated in the antrum. On average, approximately half of antral glands are colonized. Within individual antral glands H pylori microcolonies preferentially form in the mid-glandular region (Figure 1A and B). This region is known to contain proliferating progenitor cells. Some bacteria also reach the base of the antral glands, where stem cells are known to reside (Figure 1A and C). We mapped and quantified the invasion depth of H pylori into antral glands and found that the majority of infected glands (56.8% ± 2.8%) were colonized by bacteria that reached the midglandular progenitor cell compartment. About one-third of colonized glands (32.0% ± 2.9%) had H pylori in the base of the glands (Supplementary Figure 1). In some mice, H pylori also colonized the glands of the corpus. Here, the bacteria were also preferentially found in

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the neck/isthmus where immature cells reside (Figure 1D and E). To confirm that H pylori microcolonies form directly on proliferating gastric progenitor cells, we searched for host cells undergoing mitosis (Figure 1B and E, Supplementary Video 1), and visualized cells undergoing DNA synthesis by incorporation of 5-ethynyl-20 -deoxyuridine into cell nuclei (Figure 1F).

H pylori Colonize the Progenitor and Stem Cell Regions Within Human Gastric Glands We analyzed full-thickness gastric resection specimens from H pylori–infected individuals that underwent bariatric surgery using 3D confocal microscopy (Figure 2A–E, and Supplementary Figure 2). Comparison of histologic sections with 3D confocal reconstructions showed that conventional methods underestimate the level of bacterial colonization, and miss bacterial microcolonies deep in the gastric glands (Supplementary Figure 2). As is commonly described in the literature, we found the majority of bacteria within the gastric mucus and attached to surface epithelial cells at the entrance to the gastric pits (Supplementary Figure 2A–G). However, we were also able to visualize discrete bacterial microcolonies in the neck region and the base of the human glands (Figure 1, Supplementary Figure 1H–K). Further, we documented that H pylori microcolonies form directly over epithelial tight junctions (Supplementary Figure 2, Supplementary Video 2). As in the murine model, H pylori form microcolonies in the proliferative zone of the human gastric glands, where they can be found in direct contact with mitotic progenitor cells (Figure 1B, Supplementary Figure 2H, Supplementary Figure 3D).

Regional Pathology Correlates With the Presence of Gland-Associated H pylori We noticed that all C57BL/6 animals had conspicuous gland-associated H pylori microcolonies in the antral glands, but only some mice had microcolonies in the glands of the corpus. This difference was pronounced in mice from 2 different commercial sources. We performed quantitative 3D volumetric analyses of bacterial microcolonies in mice from Jackson Laboratories (Bar Harbor, ME) vs those from Charles River (Wilmington, MA) (Supplementary Figure 4A and B). In mice from Jackson Laboratories, only antral glands were colonized, while both antral and corpus glands were colonized in mice from Charles River (Supplementary Figure 4B). This difference in bacterial distribution was not due to a difference in total bacterial counts (Supplementary Figure 4C), and it did not reflect regional differences in bacterial loads in the stomachs, because colony-forming unit levels in the antrum and corpus were equivalent regardless of the presence of bacteria in the glands (Supplementary Figure 4D). Confocal microscopy of whole mounts of stomachs revealed that bacteria are present in the surface mucus in both corpus and antrum in the Jackson Laboratory mice (Supplementary Figure 5), indicating that bacteria in the mucus are distributed throughout the stomach, and glandassociated bacterial microcolonies are regional.

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Figure 1. H pylori colonize the progenitor and stem cell compartments of murine gastric glands. 3D-confocal reconstructions of murine gastric mucosa stained to visualize H pylori (green), the host actin cytoskeleton (red), and nuclei (blue). (A) H pylori tissue distribution in the mouse antrum 2 weeks postinfection. Arrows point to free-swimming H pylori in the surface mucus. Gland-associated microcolonies are magnified in (B) in the proliferative zone next to a mitotic (mi) cell, and in (C) in the gland base. (D) Gland-associated H pylori in the neck of the corpus near parietal cells (asterisk) and mitotic cells (mi). The boxed region is magnified in panel E. (F) H pylori (green) in the proliferative zone in the antrum (left) and corpus (right) labeled with 5ethynyl-20 -deoxyuridine (white). Scale bars ¼ 100 mm in panels A and D and 10 mm in others.

Although we do not know which environmental factors control the differences in gland colonization of the corpus, we asked whether pathologic outcomes differ between mice with different patterns of gland-associated bacterial colonization. We compared the pathologic changes in these 2 groups after 2 months of infection with H pylori . We found that in all mice, the antrum showed hyperplasia and signs of chronic inflammation (Supplementary Figure 4E and F). However, only Charles River mice developed inflammation and hyperplasia in the corpus (Supplementary Figure 4E and F). These data suggest the hypothesis that colonization of gastric glands rather than the colonization of the lumenal

surface by H pylori precedes regional pathologic changes in the stomach. H pylori colonization of the antral glands accelerates Lgr5þ stem cell–derived gland turnover. Because hyperplastic responses occur in areas of the stomach where glands are colonized (Supplementary Figure 3), and because we observed that H pylori stably colonize and interact with progenitor and stem cells, we asked whether H pylori infection locally affects the turnover of antral Lgr5þ stem cells and their progeny.17 To compare the turnover rates in individual antral glands, we used the lineage tracing technique to mark cells

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Figure 2. H pylori colonize the progenitor and stem cell compartments of the human gastric glands. (A) Confocal reconstruction of human antral glands stained to visualize H pylori (green), the host actin cytoskeleton (red), and nuclei (blue). (B) Gland-associated H pylori near mitotic cells (mi; arrows) in the antral gland proliferative zone. (C–E) Distribution of glandassociated H pylori in human corpus glands. (E) Cross section through the entire depth of the corpus mucosa. Boxed regions show gland-associated H pylori in the neck (C) and base (D) of the corpus. Scale bars ¼ 100 mm in panels A and E and 10 mm in others.

that derive from Lgr5þ stem cells. We used Lgr5-eGFP-IRESCreERT2/Rosa26-TdTomato mice. These mice express green fluorescent protein (GFP) in Lgr5þ stem cells and can be induced to express TdTomato in cells derived from Lgr5þ stem cells by treatment with tamoxifen.17,18 We infected these mice with H pylori for various lengths of times. As with all other mouse lines, we confirmed a stable colonization of the antral glands. Corpus glands were not colonized and no hyperplasia was observed in the corpus. Previously, lineage tracing experiments have shown that Lgr5þ stem cells regenerate entire glands in the antrum every 10–14 days.19 To induce partial lineage tracing, we injected tamoxifen as a single dose 5 days before harvesting. We imaged the antrum by confocal microscopy and measured the length of tracing in each gland labeled with TdTomato. We found that after 2 months of H pylori infection, lineage tracing in the antral glands is significantly accelerated compared with glands in uninfected mice (Figure 3A and B). Histologic examination also revealed that infection induced hyperplasia and chronic inflammation in the antrum (Figure 3C and D). Because the distribution of H pylori in the antral glands is not homogeneous throughout the stomach, we tested whether direct gland colonization correlates with localized

stem cell activation. We first compared the lineage tracing in colonized glands vs glands without H pylori within infected animals and found that colonized glands have significantly higher lineage tracing compared with glands without H pylori in the same animals (Supplementary Figure 6). We then mapped the location and quantity of the bacteria in individual glands and correlated bacterial density per gland with the lineage tracing length (Figure 3E). The mapping analysis of the antral tissue revealed a significant correlation between the number of bacteria in individual glands and the height of lineage tracing of the glands (P < .0001; r ¼ 0.67; Figure 3E, Supplementary Figure 7A). Our data shows that gland-associated H pylori induce local changes in gland turnover, which results in gland hyperplasia.

Stem Cell Activation and Increased Turnover Occur Early After Infection At 2 months of infection, the hyperproliferation of antral glands is also associated with marked chronic inflammatory changes. To determine if H pylori infection induces stem cell turnover before establishment of chronic gastritis, we infected Lgr5-reporter mice for 2 weeks. At this time point,

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Figure 3. Gland-associated H pylori induce Lgr5þ stem cell-derived hyperproliferation and hyperplasia. (A) Confocal cross sections of antral glands from either uninfected (uninf) or 2- month H pylori–infected (2 mo) Lgr5-eGFP-IRES-CreERT2/ Rosa26-TdTomato mice. Lineage tracing (red) was induced 5 days before harvesting. (B) Scatter plot quantifying the lineage tracing ratio of individual glands from uninfected vs 2-month infected animals. (C) Antral glands stained with H&E from uninfected vs 2-month infected animals. (D) Antral inflammation and hyperplasia scores from uninfected vs 2-month infected animals. (E) Longitudinal section through the entire antrum of an Lgr5-eGFP-IRES-CreERT2/ Rosa26-TdTomato mouse infected for 2 months with H pylori. Gland-associated bacteria from the boxed area are magnified in the right panel. Lineage tracing (red) was induced 5 days before harvesting. Total gland height (line), gland tracing height (red), and number of bacteria per gland (green) are mapped below according to location. The scatter plot correlates the gland tracing height to the density of gland-associated H pylori combining data from 3 different animals. Results of Spearman correlation test are shown. Scale bar ¼ 100 mm.

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histologic analysis revealed mild inflammatory infiltrates and slight hyperplasia that did not yet significantly differ from uninfected control animals (Figure 4C and D). Lineage tracing analysis showed that, even as early as at 2 weeks

post infection, H pylori induced accelerated lineage tracing (Figure 4A and B), and the overall height of the glands was not yet increased significantly (Figure 4E). This indicates that even before hyperplasia is apparent, the proliferation

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rate of the glands is already increased. As with the animals infected for 2 months, at 2 weeks of infection there was a spatial correlation between the colonization of individual glands with H pylori and accelerated tracing (Figure 4F, Supplementary Figure 7B). To confirm that gland-associated bacteria increase epithelial proliferation by using alternative methods, we infected another 2 groups of mice with H pylori for 2 weeks and performed either 5-ethynyl-20 -deoxyuridine labeling for cells synthesizing DNA or phospho-histone-H3 staining for cells undergoing mitosis.20 We found a significant increase in epithelial proliferation after 2 weeks of infection using both markers. Also in accord with the lineage tracing data, the number of mitotic cells per gland was increased in glands colonized by H pylori vs noncolonized glands within the same animal (Supplementary Figure 8).

Bacterial Colonization of the Gastric Glands Is Required to Induce Stem Cell–Derived Hyperplasia We previously described that mutants in the chemotaxis regulator ChePep are able to colonize mice at similar levels as wild-type (WT) H pylori , but no gland-associated bacteria were present in the antrum.16 To determine if accelerated lineage tracing is dependent on gland-associated H pylori, we infected reporter mice with the DChePep mutant for 2 weeks. We confirmed that in these reporter mice, there is no difference in overall H pylori counts between DChePep and WT infected stomachs (Figure 5A). No bacteria were found in the antral glands in DChePep-infected animals (Figure 5C). Only a few bacteria were seen in the glands in the transitional zone between antrum and corpus in these mice (not shown). Analysis of Lgr5þ lineage tracing revealed that mice infected with the DChePep mutant did not exhibit accelerated tracing in the antrum (Figure 5B) compared with uninfected controls (Figure 5B, Supplementary Figure 9), and WT-infected mice showed accelerated tracing. This demonstrates that gland-associated H pylori are required to induce hyperproliferation in Lgr5þ stem cell–derived glands.

CagA Delivery Accelerates Proliferation of Lgr5þ Stem Cell Derived Glands To determine the role of CagA in stem cell activation, we performed lineage-tracing experiments with mice infected

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either with WT H pylori or with an isogenic DCagE mutant. The DCagE mutant is unable to deliver CagA into host cells due to a defective TFSS.21 At 2 months post infection, WT and DCagE bacteria colonized the stomachs in equal numbers (Figure 6D, right panel). Confocal microscopy confirmed the presence of DCagE H pylori in the glands, demonstrating that the delivery of CagA is not a requirement for gland colonization (Figure 6E). Quantification of tracing events in antral glands revealed a significantly higher tracing ratio in animals infected with WT as compared with the DCagE mutant (Figure 6A and B). However, the DCagE mutant still had higher tracing ratios than uninfected glands. The overall gland height was shorter in animals infected with the DCagE mutant than WT and was the same as in uninfected controls (Figure 6B). Histopathologic scoring revealed chronic inflammation in the antrum of WT and DCagE mutant animals at similar levels. However, the hyperplasia score was significantly lower in the DCagE mutant (Figure 6C and D). We mapped the tracing height and bacterial numbers in the antral glands of DCagE-infected animals (Figure 6E). We found a weaker correlation between bacterial burden in infected glands and tracing height in DCagE-infected animals as compared with WT (Figure 6E vs Figure 3E, and Supplementary Figure 7; Pearson correlation of 0.19 vs 0.67, respectively). These data suggest that the cag TFSS and CagA contribute to the hyperproliferative responses that are locally induced by gland-associated bacteria.

Lgr5þ Stem Cells Are Colonized by H pylori and Expand Upon Infection

To determine if H pylori can directly interact with Lgr5þ stem cells, we infected mice that express GFP under the Lgr5 promoter (Lgr5eGFP).17 As with other mouse lines, we found bacterial microcolonies concentrated in the midglandular compartment. However, we also documented H pylori microcolonies growing on the intercellular junctions of Lgr5þ stem cells in the bases of antral glands (Figure 7A, Supplementary Video 3). Next, we asked how H pylori infection affects the Lgr5þ stem cell compartment. We used 3D confocal reconstructions to measure the volume of the Lgr5þgland bases. After 2 months of H pylori infection, the number of Lgr5þ stem cells is significantly increased compared with uninfected animals (Figure 7B). The number of stem cells in

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Figure 4. Gland-associated H pylori induce Lgr5þ stem cell-derived hyperproliferation at 2 weeks post infection. (A) Confocal cross sections of antral glands from either uninfected (uninf) or 2-week H pylori–infected (2 wk) Lgr5-eGFP-IRES-CreERT2/ Rosa26-TdTomato mice. Lineage tracing (red) was induced 5 days before harvesting. (B) Scatter plot quantifying the lineage tracing ratio of individual glands from uninfected vs 2-week infected animals. (C) Antral glands stained with H&E from uninfected vs 2-week infected animals. (D) Antral inflammation and hyperplasia scores from uninfected vs 2-week infected animals. (E) Quantitation of the gland height of individual antral glands from uninfected, 2-week, and 2-month infected mouse stomachs. (F) Longitudinal section through the entire antrum of an Lgr5-eGFP-IRES-CreERT2/Rosa26-TdTomato mouse infected for 2 weeks with H pylori. Gland-associated bacteria from the boxed area are magnified in the right panel. Lineage tracing (red) was induced 5 days before harvesting. Total gland height (line), gland tracing height (red), and number of bacteria per gland (green) are mapped below according to location. The scatter plot correlates the gland tracing height to the density of gland-associated H pylori combining data from 3 different animals. Results of Spearman correlation test are shown. Scale bars ¼ 100 mm.

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Figure 5. DChePep mutants colonize the gastric surface mucus but not the antral glands, and fail to induce accelerated lineage tracing of Lgr5þ stem cells. (A) Viable bacterial counts (colony-forming units) from stomachs of mice infected with WT and DChePep H pylori for 2 weeks. (B) Scatter plot of the tracing ratios of individual antral glands in uninfected animals, animals infected with WT, or DChePep H pylori for 2 weeks. (C) Longitudinal section through the entire antrum of an Lgr5-eGFP-IRES-CreERT2/ Rosa26-TdTomato mouse infected for 2 weeks with DChePep H pylori. The boxed area is magnified in the right panel. No glandassociated bacteria were found. Lineage tracing (red) was induced 5 days before harvesting. Total gland height (line) and gland tracing height (red) are mapped below according to location. Scale bars ¼ 100 mm.

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animals that were infected with the DCagE mutant was increased over control, but also significantly lower than in WT infected mice (Figure 7B). To confirm an increase of Lgr5 expression in infected animals by a different method, we used single-molecule RNA in situ hybridization (RNAscope) with a probe for Lgr5 messenger RNA (mRNA). We observed expression of Lgr5 transcript exclusively at the base of the antral glands in all animals, and expression of Lgr5 mRNA increased in infected animals compared with controls (Figure 7C). The difference in Lgr5 stem cell numbers in infected animals was not evident at 2 weeks post infection. However, by quantifying the number of Lgr5þ stem cells undergoing mitosis at two weeks post infection (Supplementary Video 4), we found that Lgr5þ stem cells in infected animals had already increased their division rate, demonstrating that Lgr5þ stem cells are activated as early as 2 weeks after the start of H pylori infection (Figure 7D). We asked whether infection-induced activation of stem cells would affect their ability to form glands ex vivo using a

gastric organoid culture system. Gastric glands from uninfected and 2-month infected animals were isolated and plated in Matrigel, as described previously.17,22 The proportion of glands that grew into spherical organoids was quantified and the organoid size measured. We found that glands harvested from infected animals had a significantly higher capacity to form organoids. In addition, the size of the organoids from infected animals was significantly larger, confirming a functional activation of the stem cells upon infection (Figure 7E). To further investigate how infection with H pylori affects stem cell–related responses, we asked whether other genes associated with Lgr5-stem cells are up-regulated. We performed RNA in situ hybridization for the Wnt target gene Axin2, which has been previously shown to be expressed in the bases of antral glands similar to Lgr5.17 In addition, we performed RNA in situ hybridization for olfactomedin4. This gene has been show to be a robust marker of intestinal stem cells and has been used as a surrogate for Lgr5 stem cells.23 We find that both genes are expressed at significantly higher level in the base of

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Figure 6. H pylori mutants unable to deliver CagA (DCagE) colonize gastric glands, but their effect on Lgr5þ stem cell-derived proliferation and hyperplasia is diminished. (A) Confocal cross sections of antral glands from Lgr5-eGFP-IRES-CreERT2/ Rosa26-TdTomato mice infected for 2 months with either WT H pylori (WT) or an isogenic DCagE mutant (DCagE). Lineage tracing (red) was induced 5 days before harvesting. (B) Left: Scatter plot quantifying the lineage tracing ratio of individual glands from uninfected, WT, or DCagE-infected animals. Right: Quantification of the antral gland height in uninfected, WT, or DCagE-infected animals. (C) Cross sections of the antral glands stained with H&E from WT vs DCagE-infected animals. (D) Left: Antral inflammation and hyperplasia scores from WT vs DCagE-infected animals. Right: Viable bacterial counts (colonyforming units) from WT vs DCagE-infected stomachs harvested 2 months post infection. (E) Longitudinal section through the entire antrum of an Lgr5-eGFP-IRES-CreERT2/Rosa26-TdTomato mouse infected for 2 months with DCagE H pylori. Glandassociated bacteria from the boxed area are magnified in the right panel. Lineage tracing (red) was induced 5 days before harvesting. Total gland height (line), gland tracing height (red), and number of bacteria per gland (green) are mapped below according to location. The scatter plot correlates the gland tracing height to the density of gland-associated H pylori, combining data from 3 different animals. Results of Spearman correlation test are shown. Scale bars ¼ 100 mm.

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Figure 7. Lgr5þ stem cells are activated by H pylori infection. (A) Confocal microscopy images of the antrum from an Lgr5eGFP reporter mouse infected with H pylori for 2 weeks. Boxed region at the base of the glands is magnified in the right panel. (B) 3D confocal reconstruction of Lgr5þ cells in the base of antral glands of uninfected control and mice infected with WT H pylori for 2 months. Right: Quantification of the number of GFPexpressing cells per gland. (C) Detection of Lgr5 mRNA in the base of antral glands from uninfected and 1-month infected animals using singlemolecule RNA in situ hybridization (RNAscope). Graph shows comparison of Lgr5 mRNA signal from antral gland bases (n ¼ 3 animals per group). (D) Detection and quantification of mitoses in Lgr5þ cells. Lgr5eGFP stomachs were labeled with Phospho-Histone-H3 antibody (red). Mitotic Lgr5þ cells are identified (arrows) and quantified in uninfected vs 2-week infected mice. (E) Organoid formation assay. Left: Isolated gastric glands imaged at day 3 post plating demonstrating spherical organoids (arrow) vs cell clusters from glands that did not form organoids (arrowheads). Inset shows structure of gastric organoids by confocal microscopy stained with phalloidin (red) and 40 ,6diamidino-2-phenylindole (blue). Middle: Efficiency of organoid formation plotted as the ratio of glands that grew into organoids to total number of plated glands. Right: Scatter plot of organoid size measured in the same cultures each day for 6 days. Scale bars ¼ 100 mm.

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antral glands of infected animals (Supplementary Figure 10). We also attempted to quantify changes in gene expression in the Lgr5 stem cells by quantitative polymerase chain reaction. We isolated and sorted GFP-positive cells from Lgr5eGFP mice (Supplementary Figure 16). We found that individual cells from H pylori-infected animals have a higher GFP mean fluorescence intensity, which is driven by the Lgr5 promoter (Supplementary Figure 11). Quantitative polymerase chain reaction using primers for Lgr5 mRNA did not confirm the increase in expression, but the level of expression for Lgr5 transcript was near the detection limit. However, expression of olfactomedin4 mRNA did show a significant 6-fold increase by quantitative polymerase chain reaction in stem cells isolated from infected animals (Supplementary Figure 11).

Discussion In this study, we demonstrate that H pylori directly colonize the surface of gastric stem and progenitor cells and affect their biology. Although adhesion of H pylori to epithelial cells of the gastric mucosa has been reported consistently, most studies report bacteria attaching to superficial pit cells. Because these cells are terminally differentiated and not long-lived, an important question is how H pylori infection affects the proliferation in the glands. We found that H pylori can live deep in the gastric glands, forming gland-associated microcolonies directly on the epithelial junctions in regions where immature progenitor and long-lived stem cells reside. Gland-associated H pylori are responsible for manipulation of stem cell biology and this results in hyperplastic changes. These effects are not found in all glands, but are instead regional and correlate with the location of gland-associated bacteria. Using bacterial mutants that colonize the surface mucus, but cannot colonize the glands, we show that the colonization of the surface mucus is not sufficient to induce stem cell activation and that contact-dependent effects of the cag TFSS are important for these effects. A link between progenitor cells, H pylori, and gastric pathology has been proposed before. Visualization of bacteria in direct proximity to progenitor cells and also intracellularly was previously reported in a gnotobiotic mouse model of atrophic gastritis.24 In this model, parietal cells were genetically ablated through expression of diphtheria toxin under the H/K-ATPase promoter resulting in achlorhydria, and expansion of the progenitor cell niche in the corpus. Infection with H pylori in this model resulted in intracellular bacteria visualized within progenitor cells in the corpus.24 In our model and in human stomachs, we observe luminal extracellular H pylori microcolonies on the epithelial junctions of progenitor cells. Both models independently document that certain populations of H pylori have a tropism for the progenitor cell niche. It has been hypothesized that damage to progenitor and stem cells might be responsible for the development of gastric cancer. In support of this idea, some human studies have shown that patients with gastric cancer and H pylori

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infection show increased levels of DNA damage in Lgr5þ cells.25 In addition, increased expression of Lgr5 has been shown in early-stage intestinal type gastric cancer.26 In addition, experimental introduction of pro-oncogenic mutations into Lgr5þ stem cells results in rapid transformation in the intestine and the stomach antrum.17,27 The increased proliferation and expansion of Lgr5þ stem cells due to H pylori infection, as observed in our study, might be one mechanism by which the risk of transformation is increased. This area deserves additional study. Not only could there be a stochastically increased risk of mutations due to increased stem cell proliferation,28 but also, injection of CagA into stem cells could facilitate the acquisition of mutations by inhibiting tumor suppressors like p53,29 altering cell polarity9,10 or activating growth factor signaling pathways.11,12 In some animals, we also find that colonization of the glands of the corpus precedes and is associated with corpus hyperplasia and inflammation. Because we observe differences in gland colonization patterns in isogenic mouse lines that derive from different vendors, it is likely that environmental factors control the colonization of the corpus glands. A previous study correlated differences in H pylori–induced pathology in different vendors to differences in the stomach microbiota.30 Additional studies will be important in determining what environmental factors control the colonization of the corpus glands. Previous studies have suggested that infection with H pylori can result in epithelial hyperproliferation as a secondary effect of the chronic inflammatory response.31 We observed that activation of Lgr5þ stem occurs very early after infection, before chronic gastritis is established. The fact that stem cell activation occurs early and is spatially linked to glands colonized by bacteria suggests that direct interactions between the bacteria and host epithelial cells might drive this process. Using 3D culture models of gastric epithelium, 2 studies have recently demonstrated that infection with H pylori can induce hyperproliferation of the gastric epithelium in a CagAdependent manner.32,33 Although this provides evidence that direct interactions with the epithelium affect epithelial proliferation, indirect effects like innate inflammatory signals, and paracrine signals from stromal cells in the stem cell niche can also contribute to stem cell activation and gland hyperplasia. Experiments that combine transgenic mice lacking components of the immune system with stem cell reporter mice will be useful in determining their role in stem cell activation by H pylori and could provide new insights into the regulatory networks that control stem cell behavior. In addition, gastric epithelial organoid systems will be useful to study the effects of infection on the epithelium in the absence or presence of immune cells. H pylori is an example of a commensal pathogen. In some situations, it can function as a normal member of the microbiota, and in others it can lead to serious diseases. Our study of H pylori ’s microscopic biogeography suggests that bacterial ability to penetrate and manipulate the protected epithelial stem and progenitor cell

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compartments is a critical step in H pylori–induced gastric pathology.

Supplementary Material Note: To access the supplementary material accompanying this article, visit the online version of Gastroenterology at www.gastrojournal.org and at http://dx.doi.org/10.1053/j. gastro.2015.02.049.

References 1. Atherton JC. The pathogenesis of Helicobacter pyloriinduced gastro-duodenal diseases. Annu Rev Pathol 2006;1:63–96. 2. Ferlay J, Soerjomataram I, Ervik M, et al. Cancer Incidence and Mortality Worldwide: IARC CancerBase No. 11. Lyon, France: International Agency for Research on Cancer, 2013. Available at: http://globocan.iarc.fr. Accessed September 13, 2014. 3. Schreiber S, Konradt M, Groll C, et al. The spatial orientation of Helicobacter pylori in the gastric mucus. Proc Natl Acad Sci U S A 2004;101:5024–5029. 4. Ilver D, Arnqvist A, Ogren J, et al. Helicobacter pylori adhesin binding fucosylated histo-blood group antigens revealed by retagging. Science 1998;279:373–377. 5. Mahdavi J, Sonden B, Hurtig M, et al. Helicobacter pylori SabA adhesin in persistent infection and chronic inflammation. Science 2002;297:573–578. 6. Tan S, Tompkins LS, Amieva MR. Helicobacter pylori usurps cell polarity to turn the cell surface into a replicative niche. PLoS Pathog 2009;5:e1000407. 7. Tan S, Noto JM, Romero-Gallo J, et al. Helicobacter pylori perturbs iron trafficking in the epithelium to grow on the cell surface. PLoS Pathog 2011;7:e1002050. 8. Segal ED, Cha J, Lo J, et al. Altered states: involvement of phosphorylated CagA in the induction of host cellular growth changes by Helicobacter pylori. Proc Natl Acad Sci U S A 1999;96:14559–14564. 9. Amieva MR, Vogelmann R, Covacci A, et al. Disruption of the epithelial apical-junctional complex by Helicobacter pylori CagA. Science 2003;300:1430–1434. 10. Saadat I, Higashi H, Obuse C, et al. Helicobacter pylori CagA targets PAR1/MARK kinase to disrupt epithelial cell polarity. Nature 2007;447:330–333. 11. Suzuki M, Mimuro H, Suzuki T, et al. Interaction of CagA with Crk plays an important role in Helicobacter pyloriinduced loss of gastric epithelial cell adhesion. J Exp Med 2005;202:1235–1247. 12. Higashi H, Tsutsumi R, Muto S, et al. SH PYLORI-2 tyrosine phosphatase as an intracellular target of Helicobacter pylori CagA protein. Science 2002;295: 683–686. 13. Noto JM, Gaddy JA, Lee JY, et al. Iron deficiency accelerates Helicobacter pylori-induced carcinogenesis in rodents and humans. J Clin Invest 2013;123:479–492. 14. Ohnishi N, Yuasa H, Tanaka S, et al. Transgenic expression of Helicobacter pylori CagA induces gastrointestinal and hematopoietic neoplasms in mouse. Proc Natl Acad Sci U S A 2008;105:1003–1008.

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15. Parsonnet J, Friedman GD, Orentreich N, et al. Risk for gastric cancer in people with CagA positive or CagA negative Helicobacter pylori infection. Gut 1997; 40:297–301. 16. Howitt MR, Lee JY, Lertsethtakarn P, et al. ChePep controls Helicobacter pylori infection of the gastric glands and chemotaxis in the Epsilonproteobacteria. MBio 2011:2. 17. Barker N, Huch M, Kujala P, et al. Lgr5(þve) stem cells drive self-renewal in the stomach and build longlived gastric units in vitro. Cell Stem Cell 2010; 6:25–36. 18. Yan KS, Chia LA, Li X, et al. The intestinal stem cell markers Bmi1 and Lgr5 identify two functionally distinct populations. Proc Natl Acad Sci U S A 2012; 109:466–471. 19. Leushacke M, Ng A, Galle J, et al. Lgr5(þ) gastric stem cells divide symmetrically to effect epithelial homeostasis in the pylorus. Cell Rep 2013;5:349–356. 20. Shibata K, Inagaki M, Ajiro K. Mitosis-specific histone H3 phosphorylation in vitro in nucleosome structures. Eur J Biochem 1990;192:87–93. 21. Arnold IC, Lee JY, Amieva MR, et al. Tolerance rather than immunity protects from Helicobacter pylori-induced gastric preneoplasia. Gastroenterology 2011;140: 199–209. 22. Sato T, Stange DE, Ferrante M, et al. Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett’s epithelium. Gastroenterology 2011;141:1762–1772. 23. van der Flier LG, Haegebarth A, Stange DE, et al. OLFM4 is a robust marker for stem cells in human intestine and marks a subset of colorectal cancer cells. Gastroenterology 2009;137:15–17. 24. Oh JD, Karam SM, Gordon JI. Intracellular Helicobacter pylori in gastric epithelial progenitors. Proc Natl Acad Sci U S A 2005;102:5186–5191. 25. Uehara T, Ma D, Yao Y, et al. H. pylori infection is associated with DNA damage of Lgr5-positive epithelial stem cells in the stomach of patients with gastric cancer. Dig Dis Sci 2013;58:140–149. 26. Jang BG, Lee BL, Kim WH. Distribution of LGR5þ cells and associated implications during the early stage of gastric tumorigenesis. PLoS One 2013;8:e82390. 27. Barker N, Ridgway RA, van Es JH, et al. Crypt stem cells as the cells-of-origin of intestinal cancer. Nature 2009;457:608–611. 28. Tomasetti C, Vogelstein B. Cancer etiology. Variation in cancer risk among tissues can be explained by the number of stem cell divisions. Science 2015;347: 78–81. 29. Buti L, Spooner E, Van der Veen AG, et al. Helicobacter pylori cytotoxin-associated gene A (CagA) subverts the apoptosis-stimulating protein of p53 (ASPP2) tumor suppressor pathway of the host. Proc Natl Acad Sci U S A 2011;108:9238–9243. 30. Rolig AS, Cech C, Ahler E, et al. The degree of Helicobacter pylori-triggered inflammation is manipulated by preinfection host microbiota. Infect Immun 2013; 81:1382–1389.

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31. Eaton KA, Ringler SR, Danon SJ. Murine splenocytes induce severe gastritis and delayed-type hypersensitivity and suppress bacterial colonization in Helicobacter pylori-infected SCID mice. Infect Immun 1999; 67:4594–4602. 32. Wroblewski LE, Piazuelo MB, Chaturvedi R, et al. Helicobacter pylori targets cancer-associated apical-junctional constituents in gastroids and gastric epithelial cells. Gut 2015;64:720–730. 33. McCracken KW, Cata EM, Crawford CM, et al. Modelling human development and disease in pluripotent stemcell-derived gastric organoids. Nature 2014;516: 400–404.

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1501 1502 Received September 22, 2014. Accepted February 21, 2015. 1503 Reprint requests 1504 Address requests for reprints to: Manuel R. Amieva, XX, Stanford School of 1505 Medicine, 730 Welch Road, 2nd Floor, MC 5884 Palo Alto, California 94304. Q4 1506 e-mail: [email protected]; fax: (650) 725-8040. Q5 1507 Conflicts of interest Q6 1508 The authors disclose no conflicts. 1509 Funding 1510 This work was supported by the AGA R. Robert & Sally Funderburg Research Award in Gastric Cancer, and the Morgridge Faculty Scholar Award to Manuel 1511 R. Amieva. Michael Sigal was supported by the George Will Foundation Berlin 1512 Q7 and a fellowship from the German Research Foundation (Si 1983/1-1). 1513 1514 1515 1516 1517 1518 1519 1520 1521 1522 1523 1524 1525 1526 1527 1528 1529 1530 1531 1532 1533 1534 1535 1536 1537 1538 1539 1540 1541 1542 1543 1544 1545 1546 1547 1548 1549 1550 1551 1552 1553 1554 1555 1556 1557 1558 1559 1560 Author names in bold designate shared co-first authorship.

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Supplementary Methods H pylori Strains and Culture H pylori strain PMSS1 and isogenic deletion mutants of CagE have been described previously.1 The DChePep mutant PMSS1 strain was generated using natural recombination with genomic DNA isolated from the previously characterized DChePep mutant in strain SS1.2 The identity of the PMSS1 DChePep mutant was confirmed by genomic sequencing of the locus, by Western blot analysis with antiChePep antibodies, and by phenotypic analysis of swimming behavior. Routine culture of H pylori on Columbia blood agar plates was performed as described.3 For quantification of viable bacterial counts in infected mouse stomachs, blood agar plates were supplemented with vancomycin (90 mg/ mL), bacitracin (100 mg/mL) and nalidixic acid (10 mg/mL). For liquid cultures, H pylori were grown shaking at 120 rpm in Brucella broth (Difco) containing 10% fetal bovine serum (Gibco) in a microaerobic atmosphere at 37 C.

Antibodies and Tissue Labeling Reagents Rabbit anti–H pylori antibodies were generated against paraformaldehyde-fixed PMSS1 bacteria (Covance) and appropriate secondary antibodies (AlexaFluor-conjugated; Invitrogen) were used to visualize bacteria in gastric tissue. Rabbit-anti-phospho-Histone-H3 (Ser10) antibody (Millipore, Billerica, MA) and fluorescein isothiocyanate–conjugated rabbit-anti-phospho-Histone-H3 (Ser10) antibody (CST) were used to stain cells undergoing mitosis. Mouse-anti ZO-1 (Invitrogen) was used to stain the apical junctions. 5-Ethynyl-20 -deoxyuridine (EdU) staining was performed using the Click it Kit (Life Technologies). Ninety micrograms of EdU were injected intraperitoneally 4 hours before sacrificing mice. 40 ,6-Diamidino-2-phenylindole (DAPI) and AlexaFluor 594 phalloidin or AlexaFluor 660 phalloidin (Invitrogen) were used for visualization of the nuclei and actin cytoskeleton. Allophycocyanin-conjugated rat-antimouse epithelial-specific antigen antibody (Clone G 8.8; Biolegend) was used for fluorescence-activated cell sorting experiments.

H pylori Mouse Infection Model All procedures involving animals were approved by the Stanford University Administrative Panel on Laboratory Animal Care and were fully compliant with the US Department of Agriculture Animal Welfare Act, and our Assurance of Compliance with the Public Health Service Policy on Human Care and Use of Laboratory Animals. Five- to six-week old C57BL/6 mice were purchased from The Jackson Laboratory or Charles River Laboratories. Lgr5eGFP-IRES-CreERT2 (Lgr5eGFP)4 and Rosa26-TdTomato reporter mice were described previously.5 These mice were originally purchased from The Jackson Laboratory and were bred and housed at Stanford for several generations. All animals were maintained in autoclaved microisolator cages and provided with sterile drinking water and chow ad libitum. Animals were infected with a single oral dose of 108

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H pylori and sacrificed at indicated times. At the time of harvest, the forestomach was removed and the glandular stomach was opened along the lesser curvature and laid flat (Supplementary Figure 12). The stomach contents were removed and the tissue was divided into 2 halves along the greater curvature. For microscopy analysis, a similar longitudinal section at the midline along the greater curvature was used in all animals to minimize sampling error (Supplementary Figure 12).

Lineage Tracing Lgr5-eGFP-IRES-CreERT2 mice were crossed to Rosa26TdTomato mice to generate Lgr5-eGFP-IRES-CreERT2; Rosa26-TdTomato compound heterozygotes. Mice were injected with a single intraperitoneal dose of 4 mg/25 g tamoxifen (Cayman Chemical, Ann Arbor, MI) resuspended in corn oil (20 mg/mL) (Sigma) 5 days before sacrifice. For all infection experiments, cage littermates served as uninfected controls. Because tamoxifen has been shown to induce parietal cell loss and hyperproliferation in the corpus,6 we performed control experiments to test whether tamoxifen affects the proliferation of antral glands, or the colonization of the antral glands by H pylori. We confirmed that this single tamoxifen dose induced a 57% loss of parietal cells in the corpus (not shown). However, we found no difference in proliferative activity in the antral glands (Supplementary Figure 13). In addition, tamoxifen did not affect the distribution of gland-associated bacteria (Supplementary Figure 13). To confirm that Lgr5 cells give rise to the entire antral glands, long-term lineage tracing (21 days) was performed in uninfected animals (Supplementary Figure 14).

Viable Counts or Colony-Forming Unit Enumeration A longitudinal section of stomach tissue containing both corpus and antrum was weighed and then mechanically homogenized with a Tissue-Tearor rotor-stator homogenizer (Biospec) in Brucella Broth. In experiments to Q18 compare colonization levels of antrum and corpus, stomachs were divided into corpus and antrum, weighed, and subjected to mechanical homogenization. Serial dilutions of the homogenates were plated for colony-forming unit (CFU) enumeration and bacterial counts were expressed as CFU per gram of stomach.

Confocal Microscopy Tissue samples from human and murine stomachs were processed for confocal immunofluorescence microscopy as described previously, with minor modifications.2 Tissue samples were fixed in 2% paraformaldehyde in 100 mM phosphate buffer (pH 7.4) for 1 hour. Tissue was embedded in 4% agarose and 100- to 200-mm sections generated using a Vibratome (Leica, Solms, Germany). Tissue sections were permeabilized in phosphate-buffered saline with 3% bovine serum, 1% saponin, and 1% Triton X-100 before staining. Samples were imaged with a Zeiss LSM 700 confocal

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microscope. Quantification of H pylori within individual gastric glands was performed using Volocity Image Analysis software (Improvision), as described previously.7 Briefly, 3D reconstructed glands were manually selected and the total voxel volume (mm3) of H pylori fluorescence signal per gland was measured. The mean voxel volume of an individual bacterium was used to calculate the total number of bacteria per gland. The same measurement protocol was applied across all analyzed samples.

Quantification of the Distribution and Invasion Depth of H pylori in the Antral Glands To quantify the distribution of H pylori within the antral glands, composite images of longitudinal sections through the antrum were generated from mice infected for 2 weeks. The height of each gland was measured using ImageJ software and normalized to 100%. In each gland, the lowest point and highest points where gland-associated bacteria were found was obtained. The localization depth of H pylori within the glands, as well as the longitudinal distribution of infected glands was mapped. Using Lgr5 as marker of stem cells and EdU as a marker of replicative progenitor cells, the glands were divided into “base,” Lgr5 stem cell containing region (first 15% of the gland); “middle,” the mid-glandular compartment containing the progenitor cells (15%–45% of the gland); and “top” the upper remainder of each gland. The proportion of glands colonized at these 3 different depths was quantified and averaged using data from 4 animals. If microcolonies were found in several regions of one gland, the deepest region was counted.

Human Samples Resected stomach specimens from patients that underwent bariatric gastric surgery without a known H pylori–associated disease were collected during surgery and immediately preserved in paraformaldehyde fixative as described for murine samples. The deidentified samples were then screened for the presence of H pylori infection using immunofluorescence microscopy and polymerase chain reaction for the cagA gene. Four of 20 samples were positive for H pylori infection. Positive samples were used for analysis of bacterial localization using 3D confocal microscopy. In all positive samples, H pylori could be visualized both in the superficial mucus and in the gastric glands. The study was approved by the National Review Board for Research at Instituto Mexicano del Seguro Social. All participants were informed about the study and those willing to participate were asked to sign a consent letter.

Volumetric Analysis and Quantification of Mitoses in the Lgr5þ Stem Cells After fixation, the submucosal smooth muscle layer surrounding the antral glands was removed by blunt dissection. The tissue was then stained with DAPI and phalloidin and mounted flat with the base of the glands closest to the coverslip. The volume of the Lgr5þ cells in

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each gland was measured from 3D confocal z-stacks using Volocity software. For quantification of mitoses in the stem cells, tissue was stained with anti-phospho-Histone-H3 antibodies and the number of mitotic cells within the GFP expressing Lgr5þ cells was counted in each gland. More than 50 glands were analyzed for each sample. Mapping analysis of Lgr5 lineage tracing, gland height, and gland-associated bacteria in the antrum. One hundred to 200 mm-thick longitudinal tissue sections containing the whole antrum were obtained from Lgr5-eGFP-IRESCreERT2; Rosa26-TdTomato mice that were induced to undergo lineage tracing with tamoxifen. Images of the antrum were taken with a 10 objective and stitched into a composite of the whole antrum using Photoshop CS6 software (Adobe). Using ImageJ software, the height of each Q20 gland was measured starting with the most proximal gland that showed evidence of tracing and continuing through the antrum. Although some Lgr5þ cells are present at the base of glands in the corpus, no tracing was seen from Lgr5þ cells in the corpus. In addition to total gland height, the maximum height of gland tracing was measured for each gland. Tracing ratio was defined as the ratio of the traced part of the gland to the total gland height. Data from at least 3 animals were used to compare the differences in the gland tracing ratios between groups. Supplementary Figure 15 shows an example of how the measurements were obtained. Because not all glands express GFP under the Lgr5 promoter due to genetic mosaicism in Lgr5eGFP mice,4 glands lacking GFP expression were excluded from the analysis. To calculate the number of gland-associated H pylori per gland in composite low-magnification images taken from the whole antrum, we used the sum fluorescence intensity of H pylori staining in each gland, as shown in Supplementary Figure 15. Ten sample glands from each section were also imaged at high magnification, reconstructed in 3D using Volocity software, and the number of bacteria determined as described here. These measurements were correlated to the fluorescence intensity of the bacterial signal obtained at low magnification.

Histopathology Longitudinal sections from the same regions used for confocal microscopy were fixed in 10% buffered formalin. Samples were paraffin embedded, sectioned, and stained with H&E by the Department of Comparative Medicine at Stanford University. Slides were interpreted and scored by a veterinary pathologist (DMB) in a blinded manner.

Tissue Isolation and Fluorescence-Activated Cell Sorting Analysis For fluorescence-activated cell sorting analysis, isolation of antral glands was performed as described previously, with minor modifications.4 The stomach was opened along the lesser curvature, washed with saline solution, and the antrum dissected. The antrum was incubated for 2 hours in a buffered saline solution containing 10 mM EDTA, then

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washed in the buffered saline solution and vigorously shaken to release individual gastric glands. Isolated gastric glands were visualized by differential interference contrast microscopy and then resuspended in TrypleExpress (Gibco) and Accumax cell dissociation solution (Innovative Cell Technologies, San Diego, CA). After incubation at 37 C for 15 minutes, serum was added to inactivate the dissociation enzymes, and after another incubation period of 30 minutes cells were collected by centrifugation, resuspended in media containing 10% fetal bovine serum, and filtered through a 40-mM mesh. Cells were analyzed for enhanced GFP expression by flow cytometry (BD FACSAria II Flow Cytometer). Single viable epithelial cells were gated by forward scatter and pulse-width parameters. Live cells were gated for negative DAPI staining. Allophycocyaninconjugated antibodies against epithelial-specific antigen were used for labeling of epithelial cells. A detailed description of the gating strategy is shown in Supplementary Figure 16.

Quantitative Reverse Transcription Polymerase Chain Reaction Lgr5-GFP antral epithelial cells were fluorescenceactivated cell sorted to >95% purity, centrifuged, and resuspended in Trizol-LS (Invitrogen). Total cellular RNA was purified using the PureLink RNA Micro kit (Invitrogen) and eluted in water. RNA was then reverse transcribed using gene specific primers. RNA (1.5 mL) was combined with 5 mL 2 CellsDirect PCR mix (Invitrogen), 0.1 mL (2 U) SuperaseIN RNAse inhibitor (Applied Biosystems, Foster City, CA), 1 mL SuperScript III RT/Platinum Taq (Invitrogen), and 2.5 mL of a mixture of pooled TaqMan assays (Applied Biosystems, Carlsbad, CA), containing each assay at 1:100 dilution to a final concentration of 0.2 (Mm01320260_m1 and Mm01320261_m1 for Olfm4, Mm00438890_m1 for Lgr5 and Mm00607939_s1 for Gapdh). Reverse transcription was performed for 30 minutes at 50 C, and the reverse transcriptase was heat-inactivated for 2 minutes at 94 C. Next, the complementary DNA was preamplified for 15 cycles (each cycle: 95 C for 15 seconds, 60 C for 4 minutes). The preamplified complementary DNA was then diluted 1:5 in water. The real-time polymerase chain reactions were conducted on an ABI7900HT Thermocycler using TaqMan assays (Applied Biosystems) and the default amplification program in SDS 2.4 (Applied Biosystems) for select genes in 10 mL reactions, using 1 mL diluted preamplified complementary DNA as a template for each reaction, 3.5 mL water, 0.5 mL TaqMan assay (supplied as 20), and 5 mL 2 universal TaqMan master mix (Applied Biosystems). Reactions were done in triplicate, and fold changes were calculated using DDCt, normalizing to glyceraldehyde-3-phosphate dehydrogenase.

Gastric Organoid Culture Isolation of antral glands from uninfected and 2-month H pylori–infected mouse stomachs was performed as described previously, with minor modifications.4,8 Antrum

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tissue was incubated for 2 hours in a buffered saline solution containing 5 mM EDTA to dissociate gastric glands. Isolated gastric glands were mixed with 25 mL Matrigel (BD) and plated in 48-well plates. After polymerization of Matrigel, gastric culture medium (advanced Dulbecco’s modified Eagle medium/F12 supplemented with B27, N2, n-Acetyl-cysteine (Invitrogen), penicillin/streptomycin and gastrin (10 nM; Sigma-Aldrich) containing growth factors (50 ng/mL epidermal growth factor, 1 mg/mL R-spondin1, 100 ng/mL Noggin, 100 ng/mL fibroblast growth factor 10, and Wnt3A-conditioned media) was overlaid. For the first 2 days after seeding, the media was also supplemented with 10 mM Y-27632 (Sigma Aldrich) and CHIR99021 (250 nM; Stemgent). The medium supplemented with growth factors Q21 was changed every 2 days. The organoid cultures were photographed daily for 6 days and the size measured using ImageJ software (available at: http://imagej.nih.gov/ij/). At day 3 postplating, the efficiency of organoid formation was measured, plated glands were photographed and counted. Some of the glands grew into spherical organoids and glands that did not form organoids appeared as cell clusters. The ratio between organoid number and total glands was calculated.

RNA In Situ Hybridization Tissue sections cut at 5-mm thickness were processed for RNA in situ detection using the RNAscope Detection Kit according to manufacturer instructions (Advanced Cell Diagnostics, Hayward, CA).9 RNAscope probes used were: Lgr5 (probe region: 2165-3082); Axin2 (probe region: 3301287); Olfm4 (probe region: 251043). The probes were detected using the Fast Red detection reagent. Three to 4 samples were analyzed per condition. Images of bases of all antral glands in the samples were taken at high magnification (63 objective). Using ImageJ software, the Fast Red signal was selected by thresholding and expressed as pixels per high power field. Data are presented as mean ± SEM.

Statistics Mean values and SEs were calculated from at least 3 independent experiments. Statistical analysis was performed using GraphPad Prism software (GraphPad Software, La Jolla, CA). Student t test, Mann-Whitney U, analysis of variance, or Spearman correlation tests were used as indicated. A P value .05 was considered significant (*P < .05; **P < .01: ***P < .001; ****P < .0001; NS, not significant; P > .05).

Supplementary References 1. Arnold IC, Lee JY, Amieva MR, et al. Tolerance rather than immunity protects from Helicobacter pylori-induced gastric preneoplasia. Gastroenterology 2011;140: 199–209. 2. Howitt MR, Lee JY, Lertsethtakarn P, et al. ChePep controls Helicobacter pylori infection of the gastric glands and chemotaxis in the Epsilonproteobacteria. MBio 2011;2(4).

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3. Salama NR, Otto G, Tompkins L, et al. Vacuolating cytotoxin of Helicobacter pylori plays a role during colonization in a mouse model of infection. Infect Immun 2001;69:730–736. 4. Barker N, Huch M, Kujala P, et al. Lgr5(þve) stem cells drive self-renewal in the stomach and build longlived gastric units in vitro. Cell Stem Cell 2010; 6:25–36. 5. Yan KS, Chia LA, Li X, et al. The intestinal stem cell markers Bmi1 and Lgr5 identify two functionally distinct populations. Proc Natl Acad Sci U S A 2012; 109:466–471. 6. Huh WJ, Khurana SS, Geahlen JH, et al. Tamoxifen induces rapid, reversible atrophy, and metaplasia in

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mouse stomach. Gastroenterology 2012;142:21–24 e7. 7. Noto JM, Gaddy JA, Lee JY, et al. Iron deficiency accelerates Helicobacter pylori-induced carcinogenesis in rodents and humans. J Clin Invest 2013;123:479–492. 8. Sato T, Stange DE, Ferrante M, et al. Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett’s epithelium. Gastroenterology 2011;141:1762–1772. 9. Wang F, Flanagan J, Su N, et al. RNAscope: a novel in situ RNA analysis platform for formalin-fixed, paraffinembedded tissues. J Mol Diagn 2012;14:22–29. Author names in bold designate shared co-first authorship.

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Supplementary Figure 1. Analysis of H pylori localization in antral glands. (A) Composite confocal microscopy image of a longitudinal section through the antrum from a mouse infected with H pylori for 2 weeks; (B) boxed region from panel A; and (C) high magnification of the boxed region from panel B. (D) Map of bacterial localization within each individual gland from the image in panel A. Gland height was normalized and expressed as 100% for each gland. Each green bar of the graph in panel D starts at the lowest region bacteria are detected and ends at the highest point where gland-associated bacteria are found. Using Lgr5 as marker of stem cells and EdU as a marker of replicative progenitor cells, the glands were divided into “base,” Lgr5 stem cell containing region (first 15% of the gland); “middle,” the mid-glandular compartment containing the progenitor cells (15%–45% of the gland); and “top,” the upper remainder of each gland. (E) Proportion of glands colonized to different depths was quantified and averaged for a total of 4 animals. If microcolonies were found in several regions of one gland, the deepest region was counted. The proportion of colonized glands that had bacteria reaching the base (green, 32.0% ± 2.9%) and middle proliferative compartment (blue, 56.8% ± 2.8%), and glands where bacteria were exclusively found in the top compartment (red, 11.2% ± 1.9%) were plotted.

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Supplementary Figure 2. Colonization niches of H pylori in the human stomach can be visualized using 3D confocal microscopy. To compare standard histopathologic techniques for identification of H pylori with 3D confocal microscopy, we imaged the same human sample using 3 different techniques. (A) Low magnification H&E image of a full-thickness section through the human stomach mucosa. (B) High-magnification closeup H&E image of the gastric surface pit showing lumenal bacteria (asterisk) on the surface mucus cells. (C) Low-magnification, silver-stained image of the same sample, boxed region is magnified in panel D showing bacteria in the gastric surface pit. (E–K) 3D-confocal reconstructions of human gastric mucosa from the same specimen as in panels A–D. Bacteria are visualized with anti-H pylori antibodies (green), and the tissue is counterstained with phalloidin (red) for the actin cytoskeleton, and DAPI (blue) to stain nuclei. (E–G) H pylori colonizing the surface of the stomach: (E) H pylori free-swimming in the mucus layer. (F) Reconstruction of the lumenal surface of the stomach showing H pylori preferentially adhered to the intercellular junctions, (G) H pylori microcolonies in a surface pit region similar to panels B and D. (H–K) H pylori visualized deep in human gastric glands. (H) H pylori in the midglandular region of an antral gland next to a mitotic cell (mi) stained with anti-phospho-Histone-H3 antibodies. (I) H pylori microcolony deep in an oxyntic gland. (J) H pylori occasionally found in canaliculi of parietal cells. (K) H pylori in the base of a gastric gland. Scale bars ¼ 100 mm in panels A and C and 10 mm in the rest. p, parietal cell; sm, smooth muscle.

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Supplementary Figure 3. H pylori form microcolonies on the epithelial junctions in the human stomach glands. 3D-confocal reconstructions of human gastric tissue. Bacteria are visualized with anti-H pylori antibodies (red), and the tight junctions stained with anti–ZO-1 antibody (green), nuclei are stained with DAPI (blue). (A) Distinct H pylori microcolonies (asterisk) can be found at different depths on the epithelial junctions in the lumen of a gastric gland. (B, C) The same bacterial microcolonies are visualized from the lumenal side (B) or from the inside of the cell (C). Arrowheads point to regions where the bacteria are directly over the epithelial junctions; see also Supplementary Video 2. (D) Example of an H pylori microcolony next to a mitotic cell (mi). (E) Example of an H pylori microcolony growing on cell junctions in the base of a human gland. Scale bars ¼ 1 mm in (D) and 10 mm in other images.

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Supplementary Figure 4. Regional colonization of the glands by H pylori correlates with local pathology. (A, B) Volumetric analysis of bacteria in gastric glands of mice from different commercial vendors. (A) Example of a confocal 3D reconstruction of a gland stained with anti–H pylori (green), phalloidin (red), and DAPI (blue). A cluster of bacteria is seen in the lumen of the gland (left image). The bacterial signal was identified using an automated protocol in Volocity software and the identified voxels marked in gray (right image) and measured. (B) Using the method shown in panel A, the voxel volume of bacteria colonizing each gland was measured and the number of bacteria per gland calculated based on the average voxel volume of 1 bacterium. The number of bacteria per gland of the antrum vs the corpus is plotted for animals obtained from Jackson Laboratories (JAX) vs Charles River (CR) and infected for 2-weeks with H pylori. (C) Total viable bacterial counts (CFU) from stomachs of mice from different commercial vendors infected with H pylori for 2 weeks (left panel) and 2 months (right panel). (D) CFUs in antrum vs corpus tissue from mice infected with H pylori for 2 weeks. (E, F) Pathology score for inflammation and hyperplasia of H&E-stained stomach sections from mice infected for 2 months for the antrum (E) and corpus (F); right panels show representative H&E images. Scale bars ¼ 10 mm in panel A and 100 mm in panels E and F. Statistical comparison between the 2 groups was done using a Mann-Whitney test. FLA 5.2.0 DTD
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Supplementary Figure 5. Although gland-associated bacteria are regional, the surface mucus is colonized by H pylori throughout the stomach. Because mice from Jackson Laboratory have gland-associated bacteria exclusively in the antrum, but CFU in the tissue is equal in the antrum and corpus we imaged the surface of different regions of the stomach to determine whether H pylori colonizes the surface mucus in the corpus. Whole stomach tissues were mounted without sectioning and the lumenal surface of the stomach was imaged by 3D reconstruction confocal microscopy. Anti-H pylori antibodies (green), DAPI (blue), and phalloidin (red) were used. (A, B) Low-magnification reconstructions of the surface of the antrum and corpus showing H pylori swimming in the surface mucus. (C) High-magnification enlargement of the surface mucus in the corpus shows that bacterial signal represents individual spiral-shaped H pylori in the mucus. (D) To confirm that the region in (C) is located in the corpus, a high magnification image of the same region shown in (C) was taken deeper into the glands displaying parietal cells (p). Scale bars ¼ 100 mm in panels A and B and 10 mm in panels C and D.

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Supplementary Figure 6. Gland-associated H pylori induce increased Lgr5-lineage tracing at the level of individual glands. The tracing height of individual antral glands in the stomachs of Lgr5-eGFP-IRES-CreEr2/Rosa26-TdTomato mice infected with H pylori for 2 months was measured. Lineage tracing was induced 5 days before sacrifice by a single dose of tamoxifen. Glands colonized with H pylori were compared with noncolonized antral glands in the same animals. Gastric tissue from 3 mice was analyzed. Statistical comparison between the 2 groups was done using a MannWhitney test.

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Supplementary Figure 7. Correlation between gland-associated H pylori and Lgr5 lineage tracing in individual mice. Correlation scatter plots of Lgr5 lineage tracing vs bacteria per gland from 3 individual mice infected for either 2 months (A) or 2 weeks (B) with WT H pylori; and from mice infected for 2 months with an isogenic DCagE mutant (C). Lineage tracing was induced 5 days before sacrifice by a single dose of tamoxifen. Correlation scatter plots with the combined data from each group of 3 animals are presented in Supplementary Figures 3E, 4F, and 6E.

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Supplementary Figure 8. H pylori–induced hyperproliferation is mediated by gland-associated bacteria. (A) Confocal microscopy image of uninfected vs H pylori–infected mouse antrum stained for EdU to detect actively replicating cells (white). The number of positive cells per gland was counted. Data from 3 animals are shown (right panel). (B) Confocal microscopy images of uninfected vs H pylori–infected mouse antrum stained for phospho-Histone-H3 (white) to detect mitotic figures. The number of positive cells per gland was counted and data from 3 animals are shown (right panel). (C) Confocal microscopy images of mouse antrum infected for 2 weeks with H pylori and stained for phospho-Histone-H3 (white) and H pylori (green). Regions in the antrum without gland-associated H pylori and with gland-associated H pylori from the same animal are shown. Quantification of the number of positive cells per gland with and without H pylori from 3 animals is shown in the right panel. Statistical comparison between the 2 groups was done using a Mann-Whitney test.

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Supplementary Figure 9. Mapping analysis of Lgr5-lineage tracing in the antrum of an uninfected Lgr5-eGFP-IRES-CreEr2; Rosa26-TdTomato compound heterozygous mouse. (A) Confocal image of a longitudinal section through the entire antrum showing lineage traced glands (dTTomato, red), nuclei (DAPI, blue), and actin (phalloidin, white). Lineage tracing was induced 5 days before sacrifice by a single dose of tamoxifen. (B) Mapping analysis of the tissue shown in panel A showing gland height and lineage tracing height (red).

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3181 3182 3183 3184 3185 3186 3187 3188 3189 3190 3191 3192 3193 3194 3195 3196 3197 3198 3199 3200 3201 3202 3203 3204 3205 3206 3207 3208 3209 3210 3211 3212 3213 3214 3215 3216 3217 3218 3219 3220 3221 3222 3223 3224 3225 3226 3227 3228 3229 3230 3231 3232 3233 3234 3235 3236 3237 3238 3239 3240

3241 3242 3243 3244 3245 3246 3247 3248 3249 3250 3251 3252 3253 3254 3255 3256 3257 3258 3259 3260 3261 3262 3263 3264 3265 3266 3267 3268 3269 3270 3271 3272 3273 3274 3275 3276 3277 3278 3279 3280 3281 3282 3283 3284 3285 3286 3287 3288 3289 3290 3291 3292 3293 3294 3295 3296 3297 3298 3299 3300

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Supplementary Figure 10. Expression of stem cell–related genes is increased by H pylori infection. In situ hybridization using probes targeting Axin2 (A) and olfactomedin4 (Olfm4) (B) mRNA was performed in the stomachs of uninfected (left) and H pylori–infected (middle) mice. Positive signal (Fast Red) was measured from high-magnification images of the bases of antral glands and expressed as number of pixels per high power field (H PYLORIF; right); data from 3–4 mice per group were analyzed; data are expressed as mean ± SEM. Statistical comparison between the 2 groups was done using a Mann-Whitney test.

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3301 3302 3303 3304 3305 3306 3307 3308 3309 3310 3311 3312 3313 3314 3315 3316 3317 3318 3319 3320 3321 3322 3323 3324 3325 3326 3327 3328 3329 3330 3331 3332 3333 3334 3335 3336 3337 3338 3339 3340 3341 3342 3343 3344 3345 3346 3347 3348 3349 3350 3351 3352 3353 3354 3355 3356 3357 3358 3359 3360

3361 3362 3363 3364 3365 3366 3367 3368 3369 3370 3371 3372 3373 3374 3375 3376 3377 3378 3379 3380 3381 3382 3383 3384 3385 3386 3387 3388 3389 3390 3391 3392 3393 3394 3395 3396 3397 3398 3399 3400 3401 3402 3403 3404 3405 3406 3407 3408 3409 3410 3411 3412 3413 3414 3415 3416 3417 3418 3419 3420

2015

Helicobacter pylori–Stem Cell Interactions

13.e16

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Supplementary Figure 11. Sorted Lgr5-GFP stem cells show a higher GFP mean fluorescence intensity and increased expression of olfactomedin4 (Olfm4). (A) Contour fluorescence-activated cell sorted plots of live, single GFP-expressing cells from Lgr5eGFP mouse antrum of an uninfected mouse and a mouse infected for 2 months with WT H pylori. (B) Quantification of the mean fluorescence intensity of single Lgr5eGFP cells. (C) Quantitative polymerase chain reaction (qPCR) for Lgr5 mRNA from sorted stem cells from uninfected and 2-month infected animals was performed. (D) qPCR for Olfm4 mRNA was determined as for panel C. The relative expression of Lgr5 (C) and Olfm4 (D) mRNA normalized to the expression of the housekeeping gene Gapdh is shown. Three animals per group were used. Statistical comparison between the 2 groups was done using a Student t test.

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3421 3422 3423 3424 3425 3426 3427 3428 3429 3430 3431 3432 3433 3434 3435 3436 3437 3438 3439 3440 3441 3442 3443 3444 3445 3446 3447 3448 3449 3450 3451 3452 3453 3454 3455 3456 3457 3458 3459 3460 3461 3462 3463 3464 3465 3466 3467 3468 3469 3470 3471 3472 3473 3474 3475 3476 3477 3478 3479 3480

3481 3482 3483 3484 3485 3486 3487 3488 3489 3490 3491 3492 3493 3494 3495 3496 3497 3498 3499 3500 3501 3502 3503 3504 3505 3506 3507 3508 3509 3510 3511 3512 3513 3514 3515 3516 3517 3518 3519 3520 3521 3522 3523 3524 3525 3526 3527 3528 3529 3530 3531 3532 3533 3534 3535 3536 3537 3538 3539 3540

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Supplementary Figure 12. Image of a dissected whole murine stomach with corpus and antrum regions identified. Region marked by the stripe was dissected and used for confocal microscopy.

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No.

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3541 3542 3543 3544 3545 3546 3547 3548 3549 3550 3551 3552 3553 3554 3555 3556 3557 3558 3559 3560 3561 3562 3563 3564 3565 3566 3567 3568 3569 3570 3571 3572 3573 3574 3575 3576 3577 3578 3579 3580 3581 3582 3583 3584 3585 3586 3587 3588 3589 3590 3591 3592 3593 3594 3595 3596 3597 3598 3599 3600

-

3601 3602 3603 3604 3605 3606 3607 3608 3609 3610 3611 3612 3613 3614 3615 3616 3617 3618 3619 3620 3621 3622 3623 3624 3625 3626 3627 3628 3629 3630 3631 3632 3633 3634 3635 3636 3637 3638 3639 3640 3641 3642 3643 3644 3645 3646 3647 3648 3649 3650 3651 3652 3653 3654 3655 3656 3657 3658 3659 3660

2015

Helicobacter pylori–Stem Cell Interactions

13.e18

Supplementary Figure 13. Tamoxifen does not affect antral proliferation and H pylori colonization of antral glands. (A, B) Representative images of phospho-Histone-H3 immunostaining (green) in the antrum of untreated (A) and tamoxifen-treated animals used for Lgr5-lineage tracing (B). Nuclei are stained with DAPI (blue), actin with phalloidin (white). (C) Plots represent the number of mitotic cells per gland in untreated and tamoxifen-treated mice (singe injection of 4 mg/25 g intraperitoneally [IP] 5 days before sacrifice, n ¼ 3 mice per group). (D) Analysis of the proportion of colonized antral glands from mice infected for 2 weeks with H pylori. Plots from untreated and mice treated with tamoxifen (singe injection of 4 mg/25 g IP 5 days before sacrifice). Data presented as mean ± SEM.

Supplementary Figure 14. Long-term Lgr5-lineage tracing confirms repopulation of entire glands by Lgr5þ stem cell progeny. Confocal microscopy images from the antrum of an uninfected Lgr5-eGFP-IRES-CreEr2; Rosa26-TdTomato compound heterozygous mouse, tamoxifen was injected 21 days before sacrifice. (A) Tissue was stained with DAPI (blue) and phalloidin (white). (B) TdTomato was visualized in the same sample (red) to confirm lineage tracing of entire antral glands. Scale bar ¼ 100 mm.

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3661 3662 3663 3664 3665 3666 3667 3668 3669 3670 3671 3672 3673 3674 3675 3676 3677 3678 3679 3680 3681 3682 3683 3684 3685 3686 3687 3688 3689 3690 3691 3692 3693 3694 3695 3696 3697 3698 3699 3700 3701 3702 3703 3704 3705 3706 3707 3708 3709 3710 3711 3712 3713 3714 3715 3716 3717 3718 3719 3720

13.e19

3721 3722 3723 3724 3725 3726 3727 3728 3729 3730 3731 3732 3733 3734 3735 3736 3737 3738 3739 3740 3741 3742 3743 3744 3745 3746 3747 3748 3749 3750 3751 3752 3753 3754 3755 3756 3757 3758 3759 3760 3761 3762 3763 3764 3765 3766 3767 3768 3769 3770 3771 3772 3773 3774 3775 3776 3777 3778 3779 3780

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Supplementary Figure 15. Example of identification and quantitative analysis of Lgr5-lineage tracing and gland-associated H pylori. (A) Longitudinal section through the entire stomach antrum of a 2-week infected Lgr5-eGFP-IRES-CreEr2; Rosa26TdTomato mouse (from Supplementary Figure 4F; scale bar ¼ 100 mm). (B–E) Boxed region from (A) is shown to demonstrate the strategy used for mapping analysis. (B) Actin signal was used to measure the height of gastric glands. (C) TdTomato signal was used to measure the tracing height in every gland. The ratio between the tracing height and gland height was expressed as tracing ratio. (D) Fluorescence intensity of H pylori signal in individual glands as selected by oval was measured. (E) The merged image.

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Q26

3781 3782 3783 3784 3785 3786 3787 3788 3789 3790 3791 3792 3793 3794 3795 3796 3797 3798 3799 3800 3801 3802 3803 3804 3805 3806 3807 3808 3809 3810 3811 3812 3813 3814 3815 3816 3817 3818 3819 3820 3821 3822 3823 3824 3825 3826 3827 3828 3829 3830 3831 3832 3833 3834 3835 3836 3837 3838 3839 3840

-

2015

Helicobacter pylori–Stem Cell Interactions

3841 3842 3843 3844 3845 3846 3847 3848 3849 3850 3851 3852 3853 3854 3855 3856 3857 3858 3859 3860 3861 3862 3863 3864 3865 3866 3867 3868 3869 3870 3871 3872 3873 3874 3875 3876 3877 3878 3879 3880 3881 3882 3883 3884 3885 3886 3887 3888 3889 3890 3891 3892 3893 3894 3895 3896 3897 3898 3899 3900

13.e20

3901 3902 3903 3904 3905 3906 3907 3908 3909 3910 3911 3912 3913 3914 3915 3916 3917 3918 3919 3920 3921 3922 3923 3924 3925 3926 3927 3928 3929 3930 3931 3932 3933 3934 3935 3936 3937 3938 3939 3940 3941 3942 3943 3944 3945 3946 3947 3948 3949 3950 3951 3952 3953 3954 3955 3956 3957 3958 3959 3960 FLA 5.2.0 DTD
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14 April 2015
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13.e21

3961 3962 3963 3964 3965 3966 3967 3968 3969 3970 3971 3972 3973 3974 3975 3976 3977 3978 3979 3980 3981 3982 3983 3984 3985 3986 3987 3988 3989 3990 3991 3992 3993 3994 3995 3996 3997 3998 3999 4000 4001 4002 4003 4004 4005 4006 4007 4008 4009 4010 4011 4012 4013 4014 4015 4016 4017 4018 4019 4020

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Supplementary Figure 16. Fluorescence-activated cell sorted gating strategy for isolation of Lgr5eGFP cells. Dissociated cells from antral glands of Lgr5eGFP mice were stained in suspension with an allophycocyanin (APC)-conjugated epithelial specific antigen (ESA) antibody and with DAPI. Single cells were identified by excluding clusters and cellular debris as shown in the first 3 plots (first row). We sequentially gated on forward scatter (FSC)-A vs side scatter (SSC)-A (first row, left), then FSC-W vs FSC-H (first row, middle) and then SSC-H vs SSC-W (first row, right). Viable cells were identified by exclusion of DAPI (second row, right). An unstained control (second row, left) was used to define the gate for the DAPI-negative population. Live, GFP-positive epithelial cells were identified by ESA and GFP signal (third row, right). The plot shows 3 populations of cells: ESA-negative, GFP-negative cells (nonepithelial cells 12.1%), ESA-positive, GFP-negative cells (epithelial cells, 85.7%), and ESA-positive, GFP-positive cells (Lgr5-GFP stem cells, 2.1%). Note that no ESA-negative, GFP-positive cell population was found, confirming that Lgr5-GFP stem cells are part of the epithelial cell compartment. The cells marked with the red gate were sorted and used for quantitative polymerase chain reaction experiments. An ESA-negative, GFP-negative 293T cell suspension control was used to define the gates (third row, left). To determine the purity of the sorted population, we reanalyzed the sorted GFPþ epithelial cells (fourth row). The purity in all experiments was >95%. Gating was done with FacsDiva software.

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14 April 2015
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4021 4022 4023 4024 4025 4026 4027 4028 4029 4030 4031 4032 4033 4034 4035 4036 4037 4038 4039 4040 4041 4042 4043 4044 4045 4046 4047 4048 4049 4050 4051 4052 4053 4054 4055 4056 4057 4058 4059 4060 4061 4062 4063 4064 4065 4066 4067 4068 4069 4070 4071 4072 4073 4074 4075 4076 4077 4078 4079 4080