Search

The small intestine is among the fastest self-renewing tissues in adult mammals. This rapid turnover is fueled by the intestinal stem cells residing in the intestinal crypt. Wnt signaling plays a pivotal role in regulating intestinal stem cell renewal and differentiation, and the dysregulation of this pathway leads to cancer formation. Several studies demonstrate that intestinal stem cells follow neutral drift dynamics, as they divide symmetrically to generate other equipotent stem cells. Competition for niche space and extrinsic signals in the intestinal crypt is the governing mechanism that regulates stemness versus cell differentiation, but the underlying molecular mechanisms are still poorly understood, and it is not yet clear how this process changes during disease. In this review, we highlight the mechanisms that regulate stem cell homeostasis in the small intestine, focusing on Wnt signaling and its regulation by RNF43 and ZNRF3, key inhibitors of the Wnt pathway. Furthermore, we summarize the evidence supporting the current model of intestinal stem cell regulation, highlighting the principles of neutral drift at the basis of intestinal stem cell homeostasis. Finally, we discuss recent studies showing how cancer cells bypass this mechanism to gain a competitive advantage against neighboring normal cells.

The current COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has completely changed human life for more than two years. Upon the emergence of this new lethal virus, multiple approaches were utilized to gain basic knowledge about its biology. Moreover, modern technologies, such as the organoid model system and next-generation sequencing, enabled us to rapidly establish strategies to tackle the disease, including vaccines and therapeutics. The recently developed organoid technology reflects human physiology more closely than other model systems. Coupled with its rapidness, high efficiency, and outstanding reliability, it has provided an opportunity to develop new drugs and understand the impact of the viral pathogen on the host. Recent findings using organoids have successfully revealed the cellular tropism of the virus in different organs and identified potential drug candidates that impact the disease. This review will summarize current achievements made with organoids in the fight against COVID-19.

Recent studies have shed light on the signaling pathways required for gastric tissue maintenance and how aberrations in these key pathways lead to gastric cancer development. Although it has been shown that the WNT pathway is important for gastric epithelial homeostasis, the identity and source of the responsible canonical WNT ligands remain unknown. Furthermore, it is unclear how gastric cancer acquires WNT niche independence - an important early step in tumorigenesis. Using human and mouse gastric organoids andin vivomouse models, we found that mesenchymal WNT2B and WNT7B maintain gastric epithelium in homeostasis. Next, mouse genetic studies and single-cell multi-omics analyses revealed that activation of MAPK signaling induces secretion of WNT7B in the epithelium itself. We further confirmed that in human gastric cancer, MAPK pathway activation through HER2 overexpression or copy number gains ofWNT2confers WNT independence. Importantly, the epithelium-intrinsic WNT expression could be therapeutically inhibited. Taken together, our results reveal that normal gastric epithelial turnover relies on WNT ligands secreted by niche mesenchymal cells, while transformation involves acquisition of a WNT secretory phenotype in the epithelium - representing a potential target for therapeutic interventions.

Tissue regeneration after injury involves the dedifferentiation of somatic cells, a natural adaptive reprogramming process that leads to the emergence of injury-responsive cells with fetal-like characteristics in the intestinal epithelium. However, there is no direct evidence that adaptive reprogramming involves a shared molecular mechanism with direct cellular reprogramming. Here, we induced dedifferentiation of intestinal epithelial cells through forced partial reprogrammingin vivousing “Yamanaka factors” (Oct4, Sox2, Klf4, and c-Myc: OSKM). The OSKM-induced dedifferentiation showed similar molecular features of intestinal regeneration, including a rapid transition from homeostatic cell types to injury-responsive-like cell types. These injury-responsive-like cells, sharing a gene signature of revival stem cells and atrophy-induced villus epithelial cells, actively assisted tissue regeneration following ionizing radiation-induced acute tissue damage. In contrast to normal intestinal regeneration, which involves epi-mesenchymal crosstalk through induction ofPtgs2(encoding Cox2) upon injury, the OSKM expression promotes the autonomous production of prostaglandin E2 via epithelialPtgs1(encoding Cox1) expression. These results indicate that prostaglandin synthesis is a common mechanism for intestine epithelial regeneration but involves a different enzyme (Ptgs1for Cox1) when partial reprogramming is directly applied to the intestinal epithelium.

Image-based lineage tracing enables tissue turnover kinetics and lineage potentials of different adult cell populations to be investigated. Previously, we reported a genetic mouse model system,Red2Onco, which ectopically expressed mutated oncogenes together with red fluorescent proteins (RFP). This system enabled the expansion kinetics and neighboring effects of oncogenic clones to be dissected. We now report Red2Flpe-SCON: a new mosaic knockout system that uses multicolor reporters to label both mutant and wild-type cells. We have developed theRed2Flpemouse line for red clone-specific Flpe expression, as well as the FRT-based SCON (ShortConditional IntrON) method to facilitate tunable conditional mosaic knockouts in mice. We used the Red2Flpe-SCON method to study Sox2 mutant clonal analysis in the esophageal epithelium of adult mice which revealed that the stem cell gene, Sox2, is not essential for adult stem cell maintenance itself, but rather for stem cell proliferation and differentiation.

The mammalian intestine is one of the most rapidly self-renewing tissues, driven by actively cycling stem cells residing at the crypt bottom1,2. Together with stromal cells, Paneth cells form a major element of the niche microenvironment that provides various growth factors to orchestrate intestinal stem cell homeostasis, such as Wnt33. With 19 family members, different Wnt ligands can selectively activate β-catenin dependent (canonical) or independent (non-canonical) signaling4,5. Here, we report that Dishevelled-associated activator of morphogenesis 1 (Daam1) and its paralogue Daam2 asymmetrically regulate canonical and non-canonical Wnt (Wnt/PCP) signaling, and their function is required for Paneth cell progenitor differentiation. We found that Daam1/2 interacts with the Wnt antagonist Rnf43, and Daam1/2 double knockout stimulates canonical Wnt signaling by preventing Rnf43-dependent endo-lysosomal degradation of the ubiquitinated Wnt receptor, Frizzled (Fzd). Moreover, single-cell RNA sequencing analysis revealed that Paneth cell differentiation is impaired by Daam1/2 depletion, as a result of defective Wnt/PCP signaling. Taken together, we identified Daam1/2 as an unexpected hub molecule coordinating both canonical and non-canonical Wnt signaling, the regulation of which is fundamental for specifying an adequate number of Paneth cells while maintaining intestinal stem cell homeostasis.

While the acquisition of cellular plasticity in adult stem cells is essential for rapid regeneration after tissue injury, little is known about the underlying mechanisms governing this process. Our data reveal the coordination of airway progenitor differentiation plasticity by inflammatory signals during alveolar regeneration. Following damage, interleukin-1β (IL-1β) signalling-dependent modulation of Jag1 and Jag2 expression in ciliated cells results in the inhibition of Notch signalling in secretory cells, which drives the reprogramming and acquisition of differentiation plasticity. We identify the transcription factor Fosl2 (also known as Fra2) for secretory cell fate conversion to alveolar type 2 cells that retain the distinct genetic and epigenetic signatures of secretory lineages. We also reveal that human secretory cells positive for KDR (also known as FLK-1) display a conserved capacity to generate alveolar type 2 cells via Notch inhibition. Our results demonstrate the functional role of an IL-1β–Notch–Fosl2 axis in the fate decision of secretory cells during injury repair, proposing a potential therapeutic target for human lung alveolar regeneration. Using human airway organoids and mouse models, Choi et al. show that an IL-1β–Notch–Fosl2 signalling axis regulates the conversion of secretory cells into alveolar type 2 cells after injury.

Chemoresistance is a major cause of treatment failure in many cancers. However, the life cycle of cancer cells as they respond to and survive environmental and therapeutic stress is understudied. In this study, we utilized a microfluidic device to induce the development of doxorubicin-resistant (DOXR) cells from triple negative breast cancer (TNBC) cells within 11 days by generating gradients of DOX and medium.In vivochemoresistant xenograft models, an unbiased genome-wide transcriptome analysis, and a patient data/tissue analysis all showed that chemoresistance arose from failed epigenetic control of the nuclear protein-1 (NUPR1)/histone deacetylase 11 (HDAC11) axis, and high Nupr1 expression correlated with poor clinical outcomes. These results suggest that the chip can rapidly induce resistant cells that increase tumor heterogeneity and chemoresistance, highlighting the need for further studies on the epigenetic control of the NUPR1/HDAC11 axis in TNBC.

Embryogenesis is supported by dynamic loops of cellular interactions. Here, we create a partial mouse embryo model to elucidate the principles of epiblast (Epi) and extra-embryonic endoderm co-development (XEn). We trigger naive mouse embryonic stem cells to form a blastocyst-stage niche of Epi-like cells and XEn-like cells (3D, hydrogel free and serum free). Once established, these two lineages autonomously progress in minimal medium to form an inner pro-amniotic-like cavity surrounded by polarized Epi-like cells covered with visceral endoderm (VE)-like cells. The progression occurs through reciprocal inductions by which the Epi supports the primitive endoderm (PrE) to produce a basal lamina that subsequently regulates Epi polarization and/or cavitation, which, in return, channels the transcriptomic progression to VE. This VE then contributes to Epi bifurcation into anterior- and posterior-like states. Similarly, boosting the formation of PrE-like cells within blastoids supports developmental progression. We argue that self-organization can arise from lineage bifurcation followed by a pendulum of induction that propagates over time.

Interactions between tumour cells and the surrounding microenvironment contribute to tumour progression, metastasis and recurrence1–3. Although mosaic analyses in Drosophila have advanced our understanding of such interactions4,5, it has been difficult to engineer parallel approaches in vertebrates. Here we present an oncogene-associated, multicolour reporter mouse model—the Red2Onco system—that allows differential tracing of mutant and wild-type cells in the same tissue. By applying this system to the small intestine, we show that oncogene-expressing mutant crypts alter the cellular organization of neighbouring wild-type crypts, thereby driving accelerated clonal drift. Crypts that express oncogenic KRAS or PI3K secrete BMP ligands that suppress local stem cell activity, while changes in PDGFRloCD81+ stromal cells induced by crypts with oncogenic PI3K alter the WNT signalling environment. Together, these results show how oncogene-driven paracrine remodelling creates a niche environment that is detrimental to the maintenance of wild-type tissue, promoting field transformation dominated by oncogenic clones. By inducing changes in surrounding tissue, mutant intestinal stem cells create an unfavourable niche environment that gives them a competitive advantage over non-mutant neighbours.

Wnt/β‐catenin signaling is an ancient pathway that regulates key aspects of embryonic development, cell differentiation, proliferation, and adult stem cell homeostasis. Work from different laboratories has shed light on the molecular mechanisms underlying the Wnt pathway, including structural details of ligand–receptor interactions. One key aspect that has emerged from multiple studies is that endocytosis of the receptor complex plays a crucial role in fine‐tuning Wnt/β‐catenin signaling. Endocytosis is a key process involved in both activation as well as attenuation of Wnt signaling, but how this is regulated is still poorly understood. Importantly, recent findings show that Wnt also regulates central metabolic pathways such as the acquisition of nutrients through actin‐driven endocytic mechanisms. In this review, we propose that the Wnt pathway displays diverse characteristics that go beyond the regulation of gene expression, through a connection with the endocytic machinery., In this review, we analyze the role that endocytosis plays in regulating Wnt signaling. We also discuss recent findings on how Wnt regulates metabolic processes, through endocytic mechanisms.

Three-dimensional organoids have been widely used for developmental and disease modeling. Organoids are derived from both adult and pluripotent stem cells. Various types are available for mimicking almost all major organs and tissues in the mouse and human. While culture protocols for stepwise differentiation and long-term expansion are well established, methods for genetic manipulation in organoids still need further standardization. In this review, we summarized different methods for organoid genetics and provide the pros and cons of each method for designing an optimal strategy.

Frequent mutation of the tumour suppressor RNF43 is observed in many cancers, particularly colon malignancies. RNF43, an E3 ubiquitin ligase, negatively regulates Wnt signalling by inducing degradation of the Wnt receptor Frizzled. In this study, we discover that RNF43 activity requires phosphorylation at a triplet of conserved serines. This phospho-regulation of RNF43 is required for zebrafish development and growth of mouse intestinal organoids. Cancer-associated mutations that abrogate RNF43 phosphorylation cooperate with active Ras to promote tumorigenesis by abolishing the inhibitory function of RNF43 in Wnt signalling while maintaining its inhibitory function in p53 signalling. Our data suggest that RNF43 mutations cooperate with KRAS mutations to promote multi-step tumorigenesis via the Wnt-Ras-p53 axis in human colon cancers. Lastly, phosphomimetic substitutions of the serine trio restored the tumour suppressive activity of extracellular oncogenic mutants. Therefore, harnessing phospho-regulation of RNF43 might be a potential therapeutic strategy for tumours with RNF43 mutations., RNF43 is frequently mutated in cancers and negatively regulates Wnt signalling. Here, the authors report that RNF43 phosphorylation at a serine triplet is required for the negative regulation of Wnt signalling and that the phosphorylation of RNF43 suppresses cancer-associated oncogenic RNF43 mutants.

Significance In the mammalian ovary, primordial germ cells maintain a genomic program associated with pluripotency until they stop proliferating, move toward oogenesis, and enter meiosis. The molecular mechanisms that enable primordial germ cells to exit pluripotency and enter meiosis in a timely manner are unclear, and their identification represents a major challenge in reproductive biology because the fertility of each individual depends on this. Evidence that cessation of germ cell proliferation is a cell-autonomous event, unrelated to the number of cell divisions, led to a search for an intrinsic timing mechanism that has long remained elusive. We describe here that WNT/β-catenin signaling regulates this timing and coordinates the transition from pluripotent to gametogenesis-competent germ cells., Germ cells form the basis for sexual reproduction by producing gametes. In ovaries, primordial germ cells exit the cell cycle and the pluripotency-associated state, differentiate into oogonia, and initiate meiosis. Despite the importance of germ cell differentiation for sexual reproduction, signaling pathways regulating their fate remain largely unknown. Here, we show in mouse embryonic ovaries that germ cell–intrinsic β-catenin activity maintains pluripotency and that its repression is essential to allow differentiation and meiosis entry in a timely manner. Accordingly, in β-catenin loss-of-function and gain-of-function mouse models, the germ cells precociously enter meiosis or remain in the pluripotent state, respectively. We further show that interaction of β-catenin and the pluripotent-associated factor POU5F1 in the nucleus is associated with germ cell pluripotency. The exit of this complex from the nucleus correlates with germ cell differentiation, a process promoted by the up-regulation of Znrf3, a negative regulator of WNT/β-catenin signaling. Together, these data identify the molecular basis of the transition from primordial germ cells to oogonia and demonstrate that β-catenin is a central gatekeeper in ovarian differentiation and gametogenesis.

The generation of conditional alleles using CRISPR technology is still challenging. Here, we introduce a Short Conditional intrON (SCON, 189 bp) that enables rapid generation of conditional alleles via one-step zygote injection. SCON has conditional intronic function in various vertebrate species and its target insertion is as simple as CRISPR/Cas9-mediated gene tagging.

The E3 ubiquitin ligases RING finger protein 43 (RNF43) and zinc and RING finger 3 (ZNRF3) have received great attention for their critical role in regulating WNT signalling during adult stem cell homeostasis. By promoting the turnover of WNT receptors, Frizzled and LRP5/6, RNF43 and ZNRF3 ensure that proper levels of WNT activity are maintained in stem cells. The molecular mechanism of RNF43/ZNRF3 activity is beginning to emerge from several recent studies, yet little is known about the regulation of RNF43/ZNRF3 at the post-translational level. A study in this issue of EMBO Reports identifies the deubiquitinating enzyme USP42 as a key regulator of WNT signalling, which acts by antagonizing the ubiquitin-dependent clearance of RNF43/ZNRF3 induced by R-spondins (Giebel et al, 2021).

Taste bud cells are renewed throughout life in a process requiring innervation. Recently, we reported that R-spondin substitutes for neuronal input for taste cell regeneration. R-spondin amplifies WNT signaling by interacting with stem-cell-expressed E3 ubiquitin ligases RNF43/ZNRF3 (negative regulators of WNT signaling) and G-protein-coupled receptors LGR4/5/6 (positive regulators of WNT signaling). Therefore, we hypothesized that RNF43/ZNRF3 may serve as a brake, controlled by gustatory neuron-produced R-spondin, for regulating taste tissue homeostasis. Here, we show that mice deficient for Rnf43/Znrf3 in KRT5-expressing epithelial stem/progenitor cells (RZ dKO) exhibited taste cell hyperplasia; in stark contrast, epithelial tissue on the tongue degenerated. WNT signaling blockade substantially reversed all these effects in RZ dKO mice. Furthermore, innervation becomes dispensable for taste cell renewal in RZ dKO mice. We thus demonstrate important but distinct functions of RNF43/ZNRF3 in regulating taste versus lingual epithelial tissue homeostasis.

Adult stem cells constantly react to local changes to ensure tissue homeostasis. In the main body of the stomach, chief cells produce digestive enzymes; however, upon injury, they undergo rapid proliferation for prompt tissue regeneration. Here, we identified p57

Whole-genome sequencing (WGS) of human tumors and normal cells exposed to various carcinogens has revealed distinct mutational patterns that provide deep insights into the DNA damage and repair processes. Although ionizing radiation (IR) is conventionally known as a strong carcinogen, its genome-wide mutational impacts have not been comprehensively investigated at the single-nucleotide level. Here, we explored the mutational landscape of normal single-cells after exposure to the various levels of IR. On average, 1 Gy of IR exposure generated ∼16 mutational events with a spectrum consisting of predominantly small nucleotide deletions and a few characteristic structural variations. In ∼30% of the post-irradiated cells, complex genomic rearrangements, such aschromoplexy, chromothripsis, and breakage-fusion-bridge cycles, were resulted, indicating the stochastic and chaotic nature of DNA repair in the presence of the massive number of concurrent DNA double-strand breaks. These mutational signatures were confirmed in the genomes of 22 IR-induced secondary malignancies. With high-resolution genomic snapshots of irradiated cells, our findings provide deep insights into how IR-induced DNA damage and subsequent repair processes operate in mammalian cells.

Wnt signaling plays pivotal roles during our entire lives, from conception to death, through the regulation of morphogenesis in developing embryos and the maintenance of tissue homeostasis in adults. The regulation of Wnt signaling occurs on several levels: at the receptor level on the plasma membrane, at the β-catenin protein level in the cytoplasm, and through transcriptional regulation in the nucleus. Several recent studies have focused on the mechanisms of Wnt receptor regulation, following the discovery that the Wnt receptor frizzled (Fzd) is a target of the ubiquitin ligases, RNF43 and ZNRF3. RNF43 and ZNRF3 are homologous genes that are mutated in several cancers. The details underlying their mechanism of action continue to unfold, while at the same time raising many new questions. In this review, we discuss advances and controversies in our understanding of Wnt receptor regulation.

Moon et al. report that antigen presentation via MHC II with coreceptor PD-L1 expressed on intestinal epithelial cells is an important contributing factor for the generation of CD4+CD8αα+ intraepithelial lymphocytes., Conventional CD4+ T cells are differentiated into CD4+CD8αα+ intraepithelial lymphocytes (IELs) in the intestine; however, the roles of intestinal epithelial cells (IECs) are poorly understood. Here, we showed that IECs expressed MHC class II (MHC II) and programmed death–ligand 1 (PD-L1) induced by the microbiota and IFN-γ in the distal part of the small intestine, where CD4+ T cells were transformed into CD4+CD8αα+ IELs. Therefore, IEC-specific deletion of MHC II and PD-L1 hindered the development of CD4+CD8αα+ IELs. Intracellularly, PD-1 signals supported the acquisition of CD8αα by down-regulating the CD4-lineage transcription factor, T helper–inducing POZ/Krüppel-like factor (ThPOK), via the Src homology 2 domain–containing tyrosine phosphatase (SHP) pathway. Our results demonstrate that noncanonical antigen presentation with cosignals from IECs constitutes niche adaptation signals to develop tissue-resident CD4+CD8αα+ IELs., Graphical Abstract

The homologous E3 ubiquitin ligases RNF43/ZNRF3 negatively regulate WNT signalling activation. Recently, both genes have been found mutated in several types of cancers. Specifically, loss-of-function mutations result in adenoma formation in mouse small intestine. However, their role in liver cancer has not been explored yet. Here we describe that hepatocyte-specific deletion of both Rnf43/Znrf3 results in altered lipid metabolism and a non-alcoholic steatohepatitis (NASH) phenotype in mouse, in the absence of exogenous fat supplementation. The effect is cell-autonomous, as evidenced by the intracellular lipid accumulation detected in mutant liver organoids. Upon chronic liver damage, Rnf43/Znrf3 deletion results in impaired hepatocyte regeneration, subsequent to an imbalance between hepatocyte differentiation and proliferation, which leads to hepatocellular carcinoma. Remarkably, hepatocellular carcinoma patients with mutations in ZNRF3 also present altered lipid metabolism and poorer survival. Our findings imply that Wnt activation through the RNF43/ZNRF3 module predisposes to liver cancer by altering the liver lipid metabolic ground-state and impairing liver regeneration, which combined, facilitate the progression towards malignancy. Our results highlight the requirement for personalized therapeutic or dietary interventions for those RNF43/ZNRF3 mutated individuals at risk of developing steatosis, NASH and/or liver cancer.

Secreted extracellular matrix components which regulate craniofacial development could be reactivated and play roles in adult wound healing. We report a patient with a loss-of-function of the secreted matricellular protein SMOC2 (SPARC related modular calcium binding 2) presenting severe oligodontia, microdontia, tooth root deficiencies, alveolar bone hypoplasia, and a range of skeletal malformations. Turning to a mouse model, Smoc2-GFP reporter expression indicates SMOC2 dynamically marks a range of dental and bone progenitors. While germline Smoc2 homozygous mutants are viable, tooth number anomalies, reduced tooth size, altered enamel prism patterning, and spontaneous age-induced periodontal bone and root loss are observed in this mouse model. Whole-genome RNA-sequencing analysis of embryonic day (E) 14.5 cap stage molars revealed reductions in early expressed enamel matrix components (Odontogenic ameloblast-associated protein) and dentin dysplasia targets (Dentin matrix acidic phosphoprotein 1). We tested if like other matricellular proteins SMOC2 was required for regenerative repair. We found that the Smoc2-GFP reporter was reactivated in adjacent periodontal tissues 4 days after tooth avulsion injury. Following maxillary tooth injury, Smoc2−/− mutants had increased osteoclast activity and bone resorption surrounding the extracted molar. Interestingly, a 10-day treatment with the cyclooxygenase 2 (COX2) inhibitor ibuprofen (30 mg/kg body weight) blocked tooth injury-induced bone loss in Smoc2−/− mutants, reducing matrix metalloprotease (Mmp)9. Collectively, our results indicate that endogenous SMOC2 blocks injury-induced jaw bone osteonecrosis and offsets age-induced periodontal decay.

SummaryTissue regeneration involves a multi-step process composed of diverse cellular hierarchies and states that are also implicated in tissue dysfunction and pathogenesis. Here, we leveraged single-cell RNA sequencing analysis in combination within vivolineage tracing and organoid models to fine-map trajectories of alveolar lineage cells during injury repair and regeneration. We identified Damage-Associated Transient Progenitors (DATPs) as a distinct AT2-lineaged population arising during alveolar regeneration. Specifically, we found that IL-1β, secreted by interstitial macrophages, primes a subset ofIl1r1+AT2 cells for conversion into DATPs, via aHif1a-mediated glycolysis pathway, that are functional mediators for mature AT1 cell differentiation. Importantly, we show that chronic inflammation mediated by IL-1β prevents differentiation into AT1 cells, leading to aberrant accumulation of DATPs and impaired alveolar differentiation. Our step-wise fine-mapping of cell fate transitions demonstrates how the inflammatory niche impedes alveolar regeneration by directing stem cell fate behavior.

Tracking the fate of individual cells and their progeny through lineage tracing has been widely used to investigate various biological processes including embryonic development, homeostatic tissue turnover, and stem cell function in regeneration and disease. Conventional lineage tracing involves the marking of cells either with dyes or nucleoside analogues or genetic marking with fluorescent and/or colorimetric protein reporters. Both are imaging-based approaches that have played a crucial role in the field of developmental biology as well as adult stem cell biology. However, imaging-based lineage tracing approaches are limited by their scalability and the lack of molecular information underlying fate transitions. Recently, computational biology approaches have been combined with diverse tracing methods to overcome these limitations and so provide high-order scalability and a wealth of molecular information. In this review, we will introduce such novel computational methods, starting from single-cell RNA sequencing-based lineage analysis to DNA barcoding or genetic scar analysis. These novel approaches are complementary to conventional imaging-based approaches and enable us to study the lineage relationships of numerous cell types during vertebrate, and in particular human, development and disease.

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.