Proliferation of the self-renewing epithelium of the gastric corpus occurs almost exclusively in the isthmus of the glands, from where cells migrate bidirectionally toward pit and base. The isthmus ...is therefore generally viewed as the stem cell zone. We find that the stem cell marker Troy is expressed at the gland base by a small subpopulation of fully differentiated chief cells. By lineage tracing with a Troy-eGFP-ires-CreERT2 allele, single marked chief cells are shown to generate entirely labeled gastric units over periods of months. This phenomenon accelerates upon tissue damage. Troy+ chief cells can be cultured to generate long-lived gastric organoids. Troy marks a specific subset of chief cells that display plasticity in that they are capable of replenishing entire gastric units, essentially serving as quiescent “reserve” stem cells. These observations challenge the notion that stem cell hierarchies represent a “one-way street.”
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•A subset of quiescent, differentiated chief cells express Troy•Troy+ chief cells can generate all differentiated lineages of the gastric epithelium•Troy+ chief cells act as “reserve” stem cells upon challenge of tissue homeostasis•Troy+ chief cells can initiate long-term in vitro cultures
Gastric chief cells expressing the marker Troy, although mature and differentiated, can re-enter the cell cycle and ultimately produce all cell lineages of the gastric epithelium, suggesting that they act as reserve stem cells.
Gastric cancer ranks as the fifth most common human malignancy and the third leading cause of cancer related deaths. Depending on tumor stage, endoscopic or surgical resection supported by ...perioperative chemotherapy is the only curative option for patients. Due to late clinical manifestation and missing reliable biomarkers, early detection is challenging and overall survival remains poor. Organoids are cell aggregates cultured in three-dimensions that grow with similar characteristics as their tissue-of-origin. Due to their self-renewal and proliferative capacity, organoids can be maintained long term in culture and expanded in many cases in an unlimited fashion. Patient-derived organoid (PDO) libraries function as living biobanks, allowing the in depth analysis of tissue specific function, development and disease. The recent successful establishment of gastric cancer PDOs opens up new perspectives for multiple translational clinical applications. Here, we review different adult stem cell derived gastric organoid model systems and focus on their establishment, phenotypic and genotypic characterizations as well as their use in predicting therapy response.
Several genetic and environmental factors increase gastric cancer (GC) risk, with Helicobacter pylori being the main environmental agent. GC is thought to emerge through a sequence of morphological ...changes that have been elucidated on the molecular level. New technologies have shed light onto pathways that are altered in GC, involving mutational and epigenetic changes and altered signaling pathways. Using various new model systems and innovative approaches, the relevance of such alterations for the emergence and progression of GC has been validated. Here, we highlight the key strategies and the resulting achievements. A major step is the characterization of epithelial stem cell behavior in the healthy stomach. These data, obtained through new reporter mouse lines and lineage tracing, enabled insights into the processes that control cellular proliferation, self-renewal, and differentiation of gastric stem cells. It has become evident that these cells and pathways are often deregulated in carcinogenesis. Second, insights into how H pylori colonizes gastric glands, directly interacts with stem cells, and alters cellular and genomic integrity, as well as the characterization of tissue responses to infection, provide a comprehensive picture of how this bacterium contributes to gastric carcinogenesis. Third, the development of stem cell– and tissue-specific reporter mice have driven our understanding of the signals and mutations that promote different types of GC and now also enable the study of more advanced, metastasized stages. Finally, organoids from human tissue have allowed insights into gastric carcinogenesis by validating mutational and signaling alterations in human primary cells and opening a route to predicting responses to personalized treatment.
The concept that tumors are maintained by dedicated stem cells, the so-called cancer stem cell hypothesis, has attracted great interest but remains controversial. Studying mouse models, we provide ...direct, functional evidence for the presence of stem cell activity within primary intestinal adenomas, a precursor to intestinal cancer. By "lineage retracing" using the multicolor Cre-reporter R26R-Confetti, we demonstrate that the crypt stem cell marker Lgr5 (leucine-rich repeat—containing heterotrimeric guanine nucleotide—binding protein—coupled receptor 5) also marks a subpopulation of adenoma cells that fuel the growth of established intestinal adenomas. These Lgr5 + cells, which represent about 5 to 10% of the cells in the adenomas, generate additional Lgr5 + cells as well as all other adenoma cell types. The Lgr5 + cells are intermingled with Paneth cells near the adenoma base, a pattern reminiscent of the architecture of the normal crypt niche.
Homeostasis of self-renewing small intestinal crypts results from neutral competition between Lgr5 stem cells, which are small cycling cells located at crypt bottoms. Lgr5 stem cells are interspersed ...between terminally differentiated Paneth cells that are known to produce bactericidal products such as lysozyme and cryptdins/defensins. Single Lgr5-expressing stem cells can be cultured to form long-lived, self-organizing crypt-villus organoids in the absence of non-epithelial niche cells. Here we find a close physical association of Lgr5 stem cells with Paneth cells in mice, both in vivo and in vitro. CD24(+) Paneth cells express EGF, TGF-α, Wnt3 and the Notch ligand Dll4, all essential signals for stem-cell maintenance in culture. Co-culturing of sorted stem cells with Paneth cells markedly improves organoid formation. This Paneth cell requirement can be substituted by a pulse of exogenous Wnt. Genetic removal of Paneth cells in vivo results in the concomitant loss of Lgr5 stem cells. In colon crypts, CD24(+) cells residing between Lgr5 stem cells may represent the Paneth cell equivalents. We conclude that Lgr5 stem cells compete for essential niche signals provided by a specialized daughter cell, the Paneth cell.
Cancer is a major health problem and a leading cause of death worldwide. Early cancer detection and continuous changes in treatment strategies have improved overall patient survival. The recent ...development of targeted drugs offers new opportunities for personalized cancer treatment. Nevertheless, individualized treatment is accompanied by the need for biomarkers predicting the response of a patient to a certain drug. One of the most promising breakthroughs in recent years that might help to overcome this problem is the organoid technology. Organoid cultures exhibit self-renewal capacity, self-organization, and long-term proliferation, while recapitulating many aspects of their primary tissue. Generated patient-derived organoid (PDO) libraries constitute "living" biobanks, allowing the in-depth analysis of tissue function, development, tumor initiation, and cancer pathobiology. Organoids can be derived from all gastrointestinal tissues, including esophageal, gastric, liver, pancreatic, small intestinal and colorectal tissues, and cancers of these tissues. PDOs are amenable to various techniques, including sequencing analyses, drug screening, targeted therapy testing, tumor microenvironment studies, and genetic engineering capabilities. In this review, we discuss the different applications of gastrointestinal organoids in basic cancer biology and clinical translation.
Background & Aims We previously established long-term culture conditions under which single crypts or stem cells derived from mouse small intestine expand over long periods. The expanding crypts ...undergo multiple crypt fission events, simultaneously generating villus-like epithelial domains that contain all differentiated types of cells. We have adapted the culture conditions to grow similar epithelial organoids from mouse colon and human small intestine and colon. Methods Based on the mouse small intestinal culture system, we optimized the mouse and human colon culture systems. Results Addition of Wnt3A to the combination of growth factors applied to mouse colon crypts allowed them to expand indefinitely. Addition of nicotinamide, along with a small molecule inhibitor of Alk and an inhibitor of p38, were required for long-term culture of human small intestine and colon tissues. The culture system also allowed growth of mouse Apc-deficient adenomas, human colorectal cancer cells, and human metaplastic epithelia from regions of Barrett's esophagus. Conclusions We developed a technology that can be used to study infected, inflammatory, or neoplastic tissues from the human gastrointestinal tract. These tools might have applications in regenerative biology through ex vivo expansion of the intestinal epithelia. Studies of these cultures indicate that there is no inherent restriction in the replicative potential of adult stem cells (or a Hayflick limit) ex vivo.
Defensins are endogenous antimicrobial peptides that protect the intestinal mucosa against bacterial invasion. It has been suggested that deficient defensin expression may underlie the chronic ...inflammation of Crohn disease (CD). The DNA copy number of the beta-defensin gene cluster on chromosome 8p23.1 is highly polymorphic within the healthy population, which suggests that the defective beta-defensin induction in colonic CD could be due to low beta-defensin–gene copy number. Here, we tested this hypothesis, using genomewide DNA copy number profiling by array-based comparative genomic hybridization and quantitative polymerase-chain-reaction analysis of the human beta-defensin 2 (
HBD-2) gene. We showed that healthy individuals, as well as patients with ulcerative colitis, have a median of 4 (range 2–10)
HBD-2 gene copies per genome. In a surgical cohort with ileal or colonic CD and in a second large cohort with inflammatory bowel diseases, those with ileal resections/disease exhibited a normal median
HBD-2 copy number of 4, whereas those with colonic CD had a median of only 3 copies per genome (
P=.008 for the surgical cohort;
P=.032 for the second cohort). Overall, the copy number distribution in colonic CD was shifted to lower numbers compared with controls (
P=.002 for both the surgical cohort and the cohort with inflammatory bowel diseases). Individuals with ≤3 copies have a significantly higher risk of developing colonic CD than did individuals with ⩾4 copies (odds ratio 3.06; 95% confidence interval 1.46–6.45). An
HBD-2 gene copy number of <4 was associated with diminished mucosal
HBD-2 mRNA expression (
P=.033). In conclusion, a lower
HBD-2 gene copy number in the beta-defensin locus predisposes to colonic CD, most likely through diminished beta-defensin expression.
The small intestinal epithelium is the most rapidly self-renewing tissue of mammals. Proliferative cells are confined to crypts, while differentiated cell types predominantly occupy the villi. We ...recently demonstrated the existence of a long-lived pool of cycling stem cells defined by
Lgr5 expression and intermingled with post-mitotic Paneth cells at crypt bottoms. We have now determined a gene signature for these Lgr5 stem cells. One of the genes within this stem cell signature is the Wnt target
Achaete scute-like 2 (
Ascl2). Transgenic expression of the Ascl2 transcription factor throughout the intestinal epithelium induces crypt hyperplasia and ectopic crypts on villi. Induced deletion of the
Ascl2 gene in adult small intestine leads to disappearance of the Lgr5 stem cells within days. The combined results from these gain- and loss-of-function experiments imply that Ascl2 controls intestinal stem cell fate.
Two types of stem cells are currently defined in small intestinal crypts: cycling crypt base columnar (CBC) cells and quiescent ‘+4’ cells. Here, we combine transcriptomics with proteomics to define ...a definitive molecular signature for Lgr5+ CBC cells. Transcriptional profiling of FACS‐sorted Lgr5+ stem cells and their daughters using two microarray platforms revealed an mRNA stem cell signature of 384 unique genes. Quantitative mass spectrometry on the same cell populations identified 278 proteins enriched in intestinal stem cells. The mRNA and protein data sets showed a high level of correlation and a combined signature of 510 stem cell‐enriched genes was defined. Spatial expression patterns were further characterized by mRNA in‐situ hybridization, revealing that approximately half of the genes were expressed in a gradient with highest levels at the crypt bottom, while the other half was expressed uniquely in Lgr5+stem cells. Lineage tracing using a newly established knock‐in mouse for one of the signature genes, Smoc2, confirmed its stem cell specificity. Using this resource, we find—and confirm by independent approaches—that the proposed quiescent/‘+4’ stem cell markers Bmi1, Tert, Hopx and Lrig1 are robustly expressed in CBC cells.
Transcriptome and proteome analyses of Lgr5‐positive intestinal cells define the signature of bona fide intestinal stem cells (ISCs) population. These results offer further insight into the nature of ISCs and will instruct further research on this therapeutically highly relevant topic.
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