Mechanisms of Field Cancerization in the Human Stomach: The Expansion and Spread of Mutated Gastric Stem Cells
2007; Elsevier BV; Volume: 134; Issue: 2 Linguagem: Inglês
10.1053/j.gastro.2007.11.035
ISSN1528-0012
AutoresStuart McDonald, Laura C. Greaves, Lydia Gutiérrez-González, Manuel Rodriguez‐Justo, Maesha Deheragoda, Simon J. Leedham, Robert W. Taylor, Chung Yin Lee, Sean Preston, Matthew J. Lovell, Toby Hunt, George Elia, Dahmane Oukrif, Rebecca Harrison, Marco Novelli, I. de G. Mitchell, David L. Stoker, Douglass M. Turnbull, Janusz Jankowski, Nicholas A. Wright,
Tópico(s)RNA modifications and cancer
ResumoBackground & Aims: How mutations are established and spread through the human stomach is unclear because the clonal structure of gastric mucosal units is unknown. Here we investigate, using mitochondrial DNA (mtDNA) mutations as a marker of clonal expansion, the clonality of the gastric unit and show how mutations expand in normal mucosa and gastric mucosa showing intestinal metaplasia. This has important implications in gastric carcinogenesis. Methods: Mutated units were identified by a histochemical method to detect activity of cytochrome c oxidase. Negative units were laser-capture microdissected, and mutations were identified by polymerase chain reaction sequencing. Differentiated epithelial cells were identified by immunohistochemistry for lineage markers. Results: We show that mtDNA mutations establish themselves in stem cells within normal human gastric body units, and are passed on to all their differentiated progeny, thereby providing evidence for clonal conversion to a new stem cell–derived unit—monoclonal conversion, encompassing all gastric epithelial lineages. The presence of partially mutated units indicates that more than one stem cell is present in each unit. Mutated units can divide by fission to form patches, with each unit sharing an indentical, mutant mtDNA genotype. Furthermore, we show that intestinal metaplastic crypts are clonal, possess multiple stem cells, and that fission is a mechanism by which intestinal metaplasia spreads. Conclusions: These data show that human gastric body units are clonal, contain multiple multipotential stem cells, and provide definitive evidence for how mutations spread within the human stomach, and show how field cancerization develops. Background & Aims: How mutations are established and spread through the human stomach is unclear because the clonal structure of gastric mucosal units is unknown. Here we investigate, using mitochondrial DNA (mtDNA) mutations as a marker of clonal expansion, the clonality of the gastric unit and show how mutations expand in normal mucosa and gastric mucosa showing intestinal metaplasia. This has important implications in gastric carcinogenesis. Methods: Mutated units were identified by a histochemical method to detect activity of cytochrome c oxidase. Negative units were laser-capture microdissected, and mutations were identified by polymerase chain reaction sequencing. Differentiated epithelial cells were identified by immunohistochemistry for lineage markers. Results: We show that mtDNA mutations establish themselves in stem cells within normal human gastric body units, and are passed on to all their differentiated progeny, thereby providing evidence for clonal conversion to a new stem cell–derived unit—monoclonal conversion, encompassing all gastric epithelial lineages. The presence of partially mutated units indicates that more than one stem cell is present in each unit. Mutated units can divide by fission to form patches, with each unit sharing an indentical, mutant mtDNA genotype. Furthermore, we show that intestinal metaplastic crypts are clonal, possess multiple stem cells, and that fission is a mechanism by which intestinal metaplasia spreads. Conclusions: These data show that human gastric body units are clonal, contain multiple multipotential stem cells, and provide definitive evidence for how mutations spread within the human stomach, and show how field cancerization develops. See editorial on page 628.We currently subscribe to the mutation and selection hypothesis of tumor development.1Cairns J. Mutation selection and the natural history of cancer.Nature. 1975; 255: 197-200Crossref PubMed Scopus (1236) Google Scholar, 2Tomlinson I. Bodmer W. Selection, the mutation rate and cancer: ensuring that the tail does not wag the dog.Nat Med. 1999; 5: 11-12Crossref PubMed Scopus (233) Google Scholar Thus, how mutations are established and spread within the human stomach become important questions, especially given the ongoing debate concerning the stem cell organization and clonal architecture of the human gastric mucosa.3Modlin I.M. Kidd M. Lye K.D. et al.Gastric stem cells: an update.Keio J Med. 2003; 52: 134-137Crossref PubMed Scopus (37) Google ScholarCentral to these considerations are our defitions of the terms to be used: here we define a tissue-specific stem cell as a cell that is capable of giving rise to all associated lineages. The human gastric mucosa is complex: a useful definition of the structural unit4Whitehead R. Gastrointestinal and Oesophageal Pathology.in: Churchill Livingston, Philadelphia1995: 15-32Google Scholar states that “body type gastric glands tend to be straight at the top and slightly coiled at the base; between three and seven glands drain through constricted necks into each gastric pit (or foveolus).” We therefore propose to call the whole unit the gastric unit, defined by the gastric glands associated with a single neck and foveolus. We reserve the term gland for those structures, numbering between 3 and 7, which empty into the constricted neck.Although it is becoming established that murine gastric units are clonal in origin, in human beings the issue is unclear. By using X-linked inactivation of the human androgen receptor gene, Nomura et al5Nomura S. Esumi H. Job C. et al.Lineage and clonal development of gastric glands.Dev Biol. 1998; 204: 124-135Crossref PubMed Scopus (72) Google Scholar, 6Nomura 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 (46) Google Scholar reported that although pyloric units were homotypic (and therefore monoclonal), 50% of the body units appeared to be heterotypic (polyclonal). This suggests that either there is a great deal of heterogeneity in the human stomach or that human androgen receptor analysis is not an appropriate tool. If a unit was clonal it would suggest that all its stem cells (to date there has been no evidence as to how many stem cells there are within the human gastric unit) were derived from a common ancestor that was able to give rise to the entire stem cell population of that gastric unit. Bjerknes and Cheng7Bjerknes M. Cheng H. Multipotential stem cells in adult mouse gastric epithelium.Am J Physiol. 2002; 283: G767-G777Crossref PubMed Scopus (134) Google Scholar have proposed that multiple stem cells are present in the murine gastric unit. If a mutation arose in a stem cell and this stem cell repopulated that gastric unit then all lineages should be derived from that stem cell. This is called monoclonal conversion. Indeed, there is evidence of this in reported cases of intestinal metaplasia (IM) of the stomach. Reis et al,8Reis C.A. David L. Correa P. et al.Intestinal metaplasia of human stomach displays distinct patterns of mucin (MUC1, MUC2, MUC5AC, and MUC6) expression.Cancer Res. 1999; 59: 1003-1007PubMed Google Scholar followed by van den Brink et al,9van den Brink G.R. Hardwick J.C. Nielsen C. et al.Sonic hedgehog expression correlates with fundic gland differentiation in the adult gastrointestinal tract.Gut. 2002; 51: 628-633Crossref PubMed Scopus (142) Google Scholar showed the presence of fundic gastric units in patients with IM that expressed both MUC5AC (a characteristic of gastric pit cells) and MUC2 (a characteristic of intestinal goblet cells). Moreover, van den Brink et al9van den Brink G.R. Hardwick J.C. Nielsen C. et al.Sonic hedgehog expression correlates with fundic gland differentiation in the adult gastrointestinal tract.Gut. 2002; 51: 628-633Crossref PubMed Scopus (142) Google Scholar also showed the co-existence of a normal foveolus related to a metaplastic gland. We would propose that these observations in fact reflect the process of monoclonal conversion of gastric units to entirely metaplastic units.In the context of gastric carcinogenesis, how mutations spread within the human stomach is a major unanswered question. There has always been a problem in identifying and monitoring individual stem cell clones in gastric units. We recently proposed that mitochondrial DNA (mtDNA) mutations can be used to mark such clones.10Greaves L.C. Preston S.L. Tadrous P.J. et al.Mitochondrial DNA mutations are established in human colonic stem cells, and mutated clones expand by crypt fission.Proc Natl Acad Sci U S A. 2006; 103: 714-719Crossref PubMed Scopus (228) Google Scholar Mitochondria are the major cellular generators of adenosine triphosphate (ATP) and cells contain multiple mitochondrial genomes. mtDNA is a simple, circular DNA of approximately 16.6 kb that encodes 13 essential proteins of the mitochondrial oxidative phosphorylation complexes, in addition to 22 ribsomal RNA (rRNAs) and 2 transfer RNAs (tRNAs). The mitochondrial genome is particularly susceptible to mutation because it has no protective histones, limited repair mechanisms, and resides in an environment of high oxidative stress.11Taylor R.W. Turnbull D.M. Mitochondrial DNA mutations in human disease.Nat Rev Genet. 2005; 6: 389-402Crossref PubMed Scopus (1277) Google Scholar Somatic mtDNA mutations occur randomly, increase with age,12Michikawa Y. Mazzucchelli F. Bresolin N. et al.Aging-dependent large accumulation of point mutations in the human mtDNA control region for replication.Science. 1999; 286: 774-779Crossref PubMed Scopus (618) Google Scholar, 13Brierley E.J. Johnson M.A. Lightowlers R.N. et al.Role of mitochondrial DNA mutations in human aging: implications for the central nervous system and muscle.Ann Neurol. 1998; 43: 217-223Crossref PubMed Scopus (254) Google Scholar and can affect all mtDNA copies within a cell (homoplasmy) or only a percentage (heteroplasmy). For a mutation to result in an observable mutated cellular phenotype, homoplasmy or a high degree of heteroplasmy must be present. We previously have shown in the human colon that mtDNA mutations occur within human colonic crypt stem cells and their progeny are able to populate entire crypts forming clonal units.14Taylor R.W. Barron M.J. Borthwick G.M. et al.Mitochondrial DNA mutations in human colonic crypt stem cells.J Clin Invest. 2003; 112: 1351-1360Crossref PubMed Scopus (437) Google Scholar Cytochrome c oxidase–negative crypts are very rare in patients younger than age 40 and we have not found any deficient gastric units in the human stomach of patients younger than this age (unpublished observations, McDonald et al, 2007).Here we show that mtDNA mutations, which result in cytochrome c oxidase deficiency, are present within human gastric units. We identify partially mutated units, which show the presence of multiple stem cells in a human gastric unit. Entirely mutated gastric units reveal the process of monoclonal conversion and clonal derivation from a single stem cell. We have shown that normal human gastric units are able to divide by fission into clonal patches, leading to fields of mutated gastric units. The gastric unit stem cell zone is thought to be located in the isthmus/neck region and it is highly probable that fission is initiated from this zone. Such expansion has obvious implications for gastric carcinogenesis, and because IM is considered to be an important premalignant condition we show here that IM crypts within the human stomach are clonal (incorporating all the major differentiated intestinal lineages), contain multiple multipotential stem cells, and can spread by crypt fission.Materials and MethodsPatientsPatients undergoing gastrectomy or gastroesophagectomy for adenocarcinoma were used in this study (n = 15). Morphologically normal gastric mucosa was taken from outside the tumor margins from each specimen (these specimens are histologically normal but because they are taken from cancer patients we cannot exclude the possibility of influence by the tumor), and frozen epithelial surface–side down on a microscope slide in liquid nitrogen–cooled isopentane. In one case, the presence of a mild atrophic gastritis was detected, allowing the reasonable identification of separate gastric units owing to the mild fibrosis in the lamina propria. Intestinal metaplastic mucosa was taken from a location adjacent to the tumor tissue. Multicenter ethical approval was sought and obtained as per the requirements of the United Kingdom Human Tissue Act (2006).Enzyme HistochemistryFrozen sections were cut at a thickness of 8 μm. Sequential cytochrome c oxidase and succinate dehydrogenase (SDH; the presence of which was used to highlight any deficiencies in cytochrome c oxidase) histochemistry was performed as previously published.14Taylor R.W. Barron M.J. Borthwick G.M. et al.Mitochondrial DNA mutations in human colonic crypt stem cells.J Clin Invest. 2003; 112: 1351-1360Crossref PubMed Scopus (437) Google Scholar Briefly, air-dried sections were incubated in cytochrome c oxidase medium containing 100 mmol/L cytochrome c, 20 μg/mL catalase, and 4 mmol/L diaminobenzidine tetrahydrochloride in 0.2 mol/L phosphate buffer, pH 7.0, all sourced from Sigma Aldrich (Poole, UK) for a maximum of 50 minutes at 37°C. Sections then were washed in phosphate-buffered saline (PBS) buffer, pH 7.4 for 3 × 5 minutes and then incubated in SDH medium (130 mmol/L sodium succinate, 200 mmol/L phenazine methosulfate, 1 mmol/L sodium azide, and 1.5 mmol/L nitroblue tetrazolium in 0.2 mol/L phosphate buffer, pH 7.0) for a maximum of 45 minutes at 37°C, or until a strong blue stain had developed. Sections again were washed in PBS for 3 × 5 minutes and dehydrated in a graded ethanol series (70%, 90%, 100%, 100%), cleared in Histoclear (Lamb Laboratory Supplies, Eastbourne, UK), and mounted with Permount (Fisher Scientific, FairLawn, NJ).ImmunohistochemistryFormalin-fixed paraffin sections (2 μm) were cut and allowed to air-dry overnight. All sections were dewaxed in xylene and rehydrated through decreasing alcohol concentrations, and then blocked for endogenous peroxidase in methanol/H2O2. Sections then were microwaved for 10 minutes in boiling sodium citrate buffer (pH 6.0) and allowed to slowly cool under running distilled water. Sections first were blocked with a serum-free protein block (DAKO, Ely, UK) for 10 minutes followed by incubating the sections with streptavidin for 15 minutes and then biotin, also for 15 minutes at room temperature (Vector Laboratories, Peterborough, UK). Primary antibodies were applied for 35 minutes at room temperature in a humid chamber. Primary antibodies used were as follows: sheep anti-human Pepsinogen 1 (1:800; Oy Medica Biochemica AN, Kauiainen, Finland) and mouse anti-human cytochrome c oxidase subunit 1 (1:250; Molecular Probes Invitrogen, Paisley, UK), H+K+ATPase (1:1000; DAKO), chromogranin A (1:50; DAKO), and trefoil factor family (TFF)-1 and TFF-2 (neat and 1:400, respectively; both in-house hybridomas). All antibodies were diluted in PBS with 5% fetal calf serum. Sections then were washed 3 × 5 minutes in PBS followed by 30 minutes incubation with appropriate secondary antibodies conjugated to biotin. After the final wash the sections were incubated with streptavidin peroxidase for another 30 minutes, washed, and developed in a solution containing 4 mmol/L diaminobenzidine and 0.2% hydrogen peroxide. Sections were dehydrated through alcohol, cleared with xylene, and mounted with DePeX. We used formalin-fixed, paraffin-embedded sections for lineage tracing rather than frozen sections because of their better resolution with the lineage-specific antibodies we selected. Taylor et al14Taylor R.W. Barron M.J. Borthwick G.M. et al.Mitochondrial DNA mutations in human colonic crypt stem cells.J Clin Invest. 2003; 112: 1351-1360Crossref PubMed Scopus (437) Google Scholar have shown previously that immunohistochemistry reliably detects cytochrome c oxidase deficiency.Gastric Unit Extraction and Whole-Mount HistochemistryA 1 × 1 cm piece of fresh gastric mucosa was used. The specimen was incubated in 0.1 mol/L dithiothreitol in PBS for 10 minutes and then transferred into a Petri dish containing Dulbecco’s modified Eagle medium (Invitrogen) and 30 mmol/L ethylenediaminetetraacetic acid (EDTA; Sigma) for 20 minutes without shaking. The epithelial layer then was dissected off under a stereo microscope (Zeiss, Welwyn Garden City, UK) and individual gastric units were teased off the epithelial strip. Extracted units then were incubated in cytochrome c medium for 3 minutes or until the brown color began to develop, transferred to SDH medium for 5 minutes (or until blue started to develop), postfixed in neutral-buffered formalin, and mounted in prewarmed Glycergel (DAKO).Isolation of DNA From Individual CellsFrozen sections (20 μm) were cut onto ultraviolet-irradiated P.A.L.M. membrane slides (P.A.L.M. Microlaser Biotechnologies, Bernried, Germany). Cells from cytochrome c oxidase–deficient or cytochrome c oxidase–normal gastric units were cut carefully into sterile, ultraviolet-irradiated, 0.5 mL P.A.L.M. tubes with adhesive caps (P.A.L.M.) using a P.A.L.M. Laser Microdissection system. After centrifugation (7000×g for 10 minutes) the cell was lysed in 14 μL of lysis buffer (50 mmol/L TRIS-HCl pH 8.5, 1 mmol/L EDTA, 0.5% Tween-20, 200 ng/mL proteinase K, all sourced from Sigma) at 55°C for 2 hours and then 95°C for 10 minutes to denature the proteinase K.mtDNA Sequencing of Individual Gastric Unit CellsThe entire mitochondrial genome was sequenced from each DNA sample extracted from the several component parts of individual gastric unit cells. The mitochondrial genome was amplified in overlapping fragments by using a series of M13-tailed oligonucleotide primer pairs as previously described.10Greaves L.C. Preston S.L. Tadrous P.J. et al.Mitochondrial DNA mutations are established in human colonic stem cells, and mutated clones expand by crypt fission.Proc Natl Acad Sci U S A. 2006; 103: 714-719Crossref PubMed Scopus (228) Google Scholar PCR products were sequenced by using BigDye Ver3.1 terminator cycle sequencing chemistries on an ABI Prism 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA) and compared directly with the revised Cambridge reference sequence by using SEQUENCE ANALYSIS and SEQSCAPE software (Applied Biosystems), as described.10Greaves L.C. Preston S.L. Tadrous P.J. et al.Mitochondrial DNA mutations are established in human colonic stem cells, and mutated clones expand by crypt fission.Proc Natl Acad Sci U S A. 2006; 103: 714-719Crossref PubMed Scopus (228) Google ScholarResultsMultiple Stem Cells Are Present in the Human Gastric UnitGastric units containing mtDNA mutations were identified by finding units that were deficient in histocytochemical cytochrome c oxidase activity and by using this as a marker of stem cell clonal expansion.10Greaves L.C. Preston S.L. Tadrous P.J. et al.Mitochondrial DNA mutations are established in human colonic stem cells, and mutated clones expand by crypt fission.Proc Natl Acad Sci U S A. 2006; 103: 714-719Crossref PubMed Scopus (228) Google Scholar Cytochrome c oxidase–deficient units were highlighted by the activity of nuclear-encoded SDH.14Taylor R.W. Barron M.J. Borthwick G.M. et al.Mitochondrial DNA mutations in human colonic crypt stem cells.J Clin Invest. 2003; 112: 1351-1360Crossref PubMed Scopus (437) Google Scholar We observed gastric units that were partially mutated to varying degrees. Figure 1A shows a cross-section of a gastric body–type unit with a single cytochrome c oxidase–deficient cell within the gland region. This is strong evidence for the presence of multiple stem cells because such units contain at least 2 different populations of stem cells, one expressing and one not expressing cytochrome c oxidase activity. The stem cell compartment thus is supporting 2 lineages: ergo at least 2 stem cells are present. The gastric unit stem cell zone is considered to be in the isthmus/neck region because bidirectional cellular migration is observed from this area.6Nomura 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 (46) Google Scholar We would therefore predict that a restricted phenotype of cytochrome c oxidase deficiency (whereby the foveolar cells above the neck are different from the base cells) should be detectable. Indeed, Figure 1B shows such a phenotype where the foveolar cells all are cytochrome c oxidase normal, but all the cells extending downward from the neck to the very base are all cytochrome c oxidase deficient. These data would suggest there are progenitor cells subsequent to the stem cells whose progeny either colonize the foveolus or the gland of the gastric unit. This is underlined by Figure 1D, which shows a unit in which only the parietal cell lineage remains wild type.Human Gastric Units Are ClonalWe also detected the presence of entirely mutated gastric units in which a mutated stem cell has expanded to take over the entire stem cell population. Figure 1C shows in cross-section a single unit, with 3 branches, that is entirely cytochrome c oxidase deficient. An entirely negative body type gastric unit in longitudinal section is shown in Figure 1E. The 3-dimensional architecture of the mucosa makes it difficult to be entirely certain from tissue sections that a unit is mutated entirely. We therefore extracted live human gastric units using EDTA and showed that normal human body–type units can become entirely mutated (Figure 1F and G). These data indicate that monoclonal coversion of the gastric unit has taken place and one cytochrome c oxidase–deficient stem cell has (stochastically) replaced all the cytochrome c oxidase–normal stem cells, resulting in a clonally deficient unit. To confirm clonality within a normal gastric unit, different regions of a unit were laser-captured and microdissected, and the entire mitochondrial genome of a segment of foveolus, neck region, and base from a cytochrome c oxidase–deficient gastric unit was sequenced and compared with a section from a neighboring cytochrome c oxidase–positive unit. Figure 2A and B are the precaptured and postcaptured tissue sections. Here we have shown that the foveolus, neck region, and base of the negative gastric unit possess the same mtDNA mutation (T>C transition at position 8181 in complex IV containing cytochrome c oxidase, Figure 2C), whereas the neighboring cytochrome c oxidase–positive crypt is wild type.Figure 2Normal human gastric units are clonal. (A) A cytochrome c oxidase–deficient gastric unit. (B) Negative cells have been captured from the foveolus (1), around the neck region (2) and base (3) of a negative unit, and also from the base (4) of a neighboring positive unit. (C) Sequencing revealed a T>C transition at position 8181, resulting in an I>T amino acid change in complex IV (cytochrome c oxidase genes).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Mutations Spread in the Stomach by Gland FissionWe also observed patches of entirely mutated gastric units. We define a patch as being 2 or more gastric units. The branching nature of body-type glands make it difficult to precisely quantify the number of glands, but Figure 3A shows a case with mild atrophic gastritis in which glands are separated by intersititial fibrosis in the lamina propria. There are 21 gastric glands that are entirely deficient in cytochrome c oxidase activity: these thus form at least 34Whitehead R. Gastrointestinal and Oesophageal Pathology.in: Churchill Livingston, Philadelphia1995: 15-32Google Scholar and, by inspection, 5 gastric units. This phenomenon could be caused by either random mutations occurring in neighboring units or by a single founder mutation occurring in a single gastric unit that then has colonized neighboring units. Direct sequencing of the entire mitochondrial genome revealed that laser capture–microdissected cells (Figure 3B) from each of these negative units contained an identical mutation (G>A transition at position 2593 in the 16S rRNA [MTRNR2] gene) (Figure 3C), which was not present in any of the surrounding cytochrome c oxidase–positive gastric units (Figure 3D). The chances of each unit possessing the same mutation by a stochastic process has been calculated previously as greater than 2.48 × 109:1 and we therefore conclude that a single, wholly mutated gastric unit has undergone multiple fission events resulting in a clonal patch.10Greaves L.C. Preston S.L. Tadrous P.J. et al.Mitochondrial DNA mutations are established in human colonic stem cells, and mutated clones expand by crypt fission.Proc Natl Acad Sci U S A. 2006; 103: 714-719Crossref PubMed Scopus (228) Google ScholarFigure 3Gland fission is a mechanism of mutation spread in the human stomach. (A) An entirely cytochrome c oxidase–deficient patch (on a laser-capture membrane slide). (B) Single cells from each gastric unit (a deficient unit indicated by a black arrow, a cytochrome c oxidase–normal gastric unit indicated by a white arrow) within the patch and their cytochrome c oxidase–positive neighboring units were laser capture–microdissected and the entire mitochondrial genome was sequenced. (C) All the cytochrome c oxidase–deficient cells contained the same 2593G>A transition mutation (black arrow), which was not detected in any of the neighboring cytochrome c oxidase–positive cells (D, white arrow). Importantly, this was the only nucleotide position at which mtDNA sequences from cytochrome c oxidase–deficient cells differed from those obtained in cytochrome c oxidase–positive cells.View Large Image Figure ViewerDownload Hi-res image Download (PPT)All Epithelial Lineages Within the Gastric Unit Are Derived From One Stem CellTo show that there is a common stem cell for all epithelial lineages contained within the gastric unit, all the differentiated lineages must be present in the same cytochrome c oxidase–deficient gastric unit(s). To show this, we performed immunohistochemistry on paraffin sections for markers of gastric cell lineages, serial to sections that had undergone cytochrome c oxidase immunohistochemistry. Figure 4A shows cytochrome c oxidase–mutated gastric body units that contain H+K+ATPase positive (+ve) (distinguishes parietal cells, Figure 4B), chromogranin A+ve (neuroendocrine cells, Figure 4C), TFF-1+ve (foveolar cells, Figure 4D), pepsinogen I+ve (chief cells and a population of mucous neck cells, Figure 4E), and TFF-2+ve (mucous neck cells, Figure 4F) cells. We conclude that all epithelial cell lineages are represented in these mutated gastric units, and, taken with the patch sequencing data, this is excellent evidence that all of the differentiated epithelial cell types within a patch originate from a common multipotential stem cell. Furthermore, the presence of lineage marker proteins within cytochrome c oxidase–deficient cells suggests that these cells still remain functional and there was no observable difference in expression of these proteins in comparison with neighboring wild-type cells.Figure 4All differentiated epithelial cell lineages in a gastric unit have a common ancestor. (A) Immunohistochemistry of a cytochrome c oxidase subunit 1–deficient patch. The brown foveolus on the left-hand side of the mutated patch belongs to a wild-type foveolus from a gastric unit out of the plane of section: looking to the right of the figure, the foveolus here is negative. The units contained the following: (B) H+K+ATPase+ve parietal cells, (C) chromogranin A+ve neuroendocrine cells, (D) a small number of TFF-1+ve foveolar cells, and (E) pepsinogen I+ve chief (and neck) cells. Note that some of the mucous neck cells also are stained, which is consistent with the Karam and Leblond concept of the chief cell being derived from the mucous neck cell.26Karam S.M. Straiton T. Hassan W.M. et al.Defining epithelial cell progenitors in the human oxyntic mucosa.Stem Cells. 2003; 21: 322-336Crossref PubMed Scopus (117) Google Scholar, 27Ramsey V.G. Doherty J.M. Chen C.C. et al.The maturation of mucus-secreting gastric epithelial progenitors into digestive-enzyme secreting zymogenic cells requires Mist1.Development. 2007; 134: 211-222Crossref PubMed Scopus (146) Google Scholar (F) TFF-2+ve mucus neck cells. (G and H) Anti-mouse and anti-sheep immunoglobulin (Ig)G–matched isotype controls. A small simple cyst is present. However, this is a common occurrence within the stomach cardia, and no other cysts in this tissue block were cytochrome c oxidase deficient.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Intestinal Metaplastic Crypts in the Human Stomach Are ClonalThe origins of IM in the human stomach presently are unknown. To date, only a few studies have investigated the clonality of IM crypts, using analysis of inactivation patterns of the X chromosome in females and by the use of chimeric mice.15Tatematsu M. Tsukamoto T. Inada K. Stem cells and gastric cancer: role of gastric and intestinal mixed intestinal metaplasia.Cancer Sci. 2003; 94: 135-141Crossref PubMed Scopus (170) Google Scholar This suggests that IM is a polyclonal disorder of the stomach. Moreover, CpG methylation analysis of nonexpressed genes indicates that methylation of multiple genes occurs independently in multiple units, each of which has its own stem cell.16Mihara M. Yoshida Y. Tsukamoto T. et al.Methylation of multiple genes in ga
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