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Epigenetic regulation of YAP is involved in the VPA-and-EPZ6438-driven regenerative response in vitro. a Expression of the YAP gene signature was overlaid on the regenerative trajectory from Hyper-organoids with and without VPA/EPZ6438 treatment shown in Fig. 3f. b The expression dynamics of YAP signature was cataloged in a pseudotime manner shown as a red line (successful reprogramming), a green line (failed reprogramming) and a blue line (pre-branch before bifurcation). Thick lines indicate the average gene expression patterns in each branch. c Volcano plot displaying the results of differential gene expression analysis performed between successful branch and failed branch. The dots representing YAP target genes are indicated as red. d Expression of the YAP gene signature was overlaid on the UMAPs shown in Fig. 3b and d, respectively. Hyper-and -VE/ENR-organoids are separated by the orange and blue dotted line. e Violin plots showing the entire range of metagene expression levels per single cell per cluster for the transcriptional programs of all clusters (left) and stem cell clusters (right) in different organoids. f Heatmap displaying H3K27me3 signals at promoter regions of YAP target genes in different organoids. g ChIP-seq (H3K27me3) and RNA-seq tracks of YAP target genes in different organoids.
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The capacity of 3D organoids to mimic physiological tissue organization and functionality has provided an invaluable tool to model development and disease in vitro. However, conventional organoid cultures primarily represent the homeostasis of self-organizing stem cells and their derivatives. Here, we established a novel intestinal organoid culture...
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... addition to injury-responsive Lgr5 + stem cells, we also found that cluster 17, which contained representative marker genes (Clu, Anxa1 and Sca1) of revival stem cells in an irradiation model, 23 was uniquely presented in Hyper-organoids ( Fig. 2b; Supplementary information, Fig. S4d-f). Notably, the transcriptomic profiles of cluster 17 resembled that of revival stem cells (referred to as SSC2c in in vivo irradiation model 23 ) from irradiated crypts (Supplementary information, Fig. S4e). Furthermore, cluster 17 was also enriched in fetal gene signatures, which represent the primitive molecular features of ...
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... addition to injury-responsive Lgr5 + stem cells, we also found that cluster 17, which contained representative marker genes (Clu, Anxa1 and Sca1) of revival stem cells in an irradiation model, 23 was uniquely presented in Hyper-organoids ( Fig. 2b; Supplementary information, Fig. S4d-f). Notably, the transcriptomic profiles of cluster 17 resembled that of revival stem cells (referred to as SSC2c in in vivo irradiation model 23 ) from irradiated crypts (Supplementary information, Fig. S4e). Furthermore, cluster 17 was also enriched in fetal gene signatures, which represent the primitive molecular features of injury-responsive stem cells in vivo (Supplementary information, Fig. S4e, g). ...
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... the transcriptomic profiles of cluster 17 resembled that of revival stem cells (referred to as SSC2c in in vivo irradiation model 23 ) from irradiated crypts (Supplementary information, Fig. S4e). Furthermore, cluster 17 was also enriched in fetal gene signatures, which represent the primitive molecular features of injury-responsive stem cells in vivo (Supplementary information, Fig. S4e, g). 22,23 Taken together, Hyper-organoids contain Lgr5 + and Clu + stem cell populations that resemble the injury responsive stem cells from the gut epithelium upon damage in vivo. ...
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... minus VPA/ EPZ6438 condition. Notably, genes that are targets of YAP signaling were upregulated in Hyper-organoids (Supplementary information, Fig. S10b, c). In consistent with these observations, we also found reduced levels of H3K27me3 at YAP target genes in Hyper-organoids as compared to that in organoids cultured in the absence of VPA/EPZ6438 (Fig. 4f, g), revealing a potential epigenetic mechanism underlying YAP-dependent intestinal ...
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... further explored the roles of Yap signaling in VPA/EPZ6438-driven regenerative response using single-cell sequencing data. Notably, we observed enrichment of YAP signaling along the regenerative trajectory of Hyper-organoids, which was absent in the failed branch without VPA/EPZ6438 treatment (Fig. 4a-c). Consistent with this observation, the upregulation of the YAP transcriptional program in Hyper-organoids, especially in stem cell clusters, was dependent on the addition of VPA and EPZ6438 (Fig. 4d, e). Fig. S11a). Importantly, the enrichment of Tead2 regulon was observed in Hyper-organoids, especially in injury-responsive Lgr5 + stem ...
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... of YAP signaling along the regenerative trajectory of Hyper-organoids, which was absent in the failed branch without VPA/EPZ6438 treatment (Fig. 4a-c). Consistent with this observation, the upregulation of the YAP transcriptional program in Hyper-organoids, especially in stem cell clusters, was dependent on the addition of VPA and EPZ6438 (Fig. 4d, e). Fig. S11a). Importantly, the enrichment of Tead2 regulon was observed in Hyper-organoids, especially in injury-responsive Lgr5 + stem cells, which was dependent on the treatment of VPA and EPZ6438 (Supplementary information, Fig. S11b-d). Moreover, organoid-forming efficiency was significantly impaired by the knockdown of Tead2 in ...
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... greatly disturbed VPA/EPZ6438-induced regenerative features whereas YAP activation could partially compensate for the roles of VPA/EPZ6438 in eliciting regenerative response in Hyperorganoids (Supplementary information, Fig. S10d). Interestingly, H3K27me3 levels in the locus of YAP target genes were significantly reduced by VPA/EPZ6438 treatment (Fig. 4f, g), which provides novel insight into the YAP-dependent mechanisms underlying the intestinal regeneration process. Collectively, these results suggested that YAP activation played an important role in regulating the regenerative response in Hyper-organoids. In addition to YAP signaling, other signalings like Wnt could also be critical ...
Citations
... In contrast to the traditional ENR culture system, the 8C culture system [LDN193189, glycogen synthase kinase 3 inhibitor XV, pexmetinib, valproic acid, EPZ6438, EGF, R-Spondin 1 conditioned medium and basic fibroblast growth factor (bFGF)], proposed by Qu et al (53), exhibited a nearly 130-fold increase in expression of the stem cell antigen-1 gene associated with organ regeneration after radiation. Additionally, the repair genes clusterin, annexin A1 and regenerating islet-derived β showed nearly 300-, 160-and 22-fold increases, respectively. ...
... Additionally, the repair genes clusterin, annexin A1 and regenerating islet-derived β showed nearly 300-, 160-and 22-fold increases, respectively. Consequently, this system significantly accelerates the restoration of the crypt structure in the intestinal epithelium following radiation injury (53). Furthermore, the Yap pathway plays a crucial role in crypt regeneration post-ionizing radiation by suppressing the Wnt signaling pathway while inducing the Egfr signaling pathway, ultimately promoting the proliferation of Lgr5 + ISCs (54). ...
... In 2017, organic technology was recognized as the Best Method of the Year in the field of life sciences by Nature Methods [11]. After over a decade of research, various organoid models have been successfully established, including the intestines [9,12,13], liver [10,14,15], pancreas [16,17], lungs [18][19][20], stomach [21][22][23], esophagus [24,25], bladder [26], kidneys [27][28][29], ovaries [30,31], mammary glands [32][33][34], prostate [35][36][37], brain [38][39][40], and heart [41,42]. Compared with traditional models, organoids can self-organize into three-dimensional structures, simulate the development process and physiological environment of in vivo organs under the regulation of exogenous growth factors, undergo multilineage differentiation, self-organize to form tissue or organ structures similar to the source tissue, and exhibit certain organ functions. ...
The liver is a major metabolic organ of the human body and has a high incidence of diseases. In recent years, the annual incidence of liver disease has increased, seriously endangering human life and health. The study of the occurrence and development mechanism of liver diseases, discovery of new therapeutic targets, and establishment of new methods of medical treatment are major issues related to the national economy and people’s livelihood. The development of stable and effective research models is expected to provide new insights into the pathogenesis of liver diseases and the search for more effective treatment options. Organoid technology is a new in vitro culture system, and organoids constructed by human cells can simulate the morphological structure, gene expression, and glucose and lipid metabolism of organs in vivo, providing a new model for related research on liver diseases. This paper reviews the latest research progress on liver organoids from the establishment of cell sources and application of liver organoids and discusses their application potential in the field of liver disease research.
... In addition, a qPCR analysis showed that the chip we constructed contained several of the most important cell types in the intestine, such as enteroendocrine cells (CHGA) and goblet cells (MUC2). Additionally, the cell types in the chip were similar to the cellular composition of intestinal organoids constructed in other studies [30]. ...
During ageing, the permeability of the intestinal barrier increases, the integrity of the intestinal barrier decreases, and the physiology of intestinal cells changes. Furthermore, intestinal inflammation and excessive oxidative stress are both likely to cause systemic diseases. Ginseng oligopeptides have a positive significant effect in terms of improving human health and delaying ageing, but their role in the ageing of the intestine has not been studied much. In our experiment, we constructed a gut-on-a-chip model and induced senescence of the chip with H2O2 so as to explore the effects of ginseng oligopeptides on the senescent intestine. The experimental results showed that ginseng oligopeptides had no obvious effects on the integrity of the intestine, including the TEER value and the expression of tight junction proteins. However, ginseng oligopeptides might have other positive effects, such as inhibiting excessive cell proliferation, promoting mucin secretion, and increasing the antioxidant capacity of the intestine, to improve intestinal health.
... Qu et al. present a groundbreaking organoid culture method centered around novel small intestinal organoids possessing regenerative capabilities for injury repair. They further introduce a novel combination of epithelial chemical small molecules designed to facilitate the restoration of intestinal epithelial tissue in a mouse model (46). This culture condition comprises eight chemical components designed to amplify the expression of regenerative signals associated with injury repair while preserving the expression of gut-specific markers. ...
The 3D culture of intestinal organoids entails embedding isolated intestinal crypts and bone marrow mesenchymal stem cells within a growth factor-enriched matrix gel. This process leads to the formation of hollow microspheres with structures resembling intestinal epithelial cells, which are referred to as intestinal organoids. These structures encompass various functional epithelial cell types found in the small intestine and closely mimic the organizational patterns of the small intestine, earning them the name “mini-intestines”. Intestinal tumors are prevalent within the digestive system and represent a significant menace to human health. Through the application of 3D culture technology, miniature colorectal organs can be cultivated to retain the genetic characteristics of the primary tumor. This innovation offers novel prospects for individualized treatments among patients with intestinal tumors. Presently established libraries of patient-derived organoids serve as potent tools for conducting comprehensive investigations into tissue functionality, developmental processes, tumorigenesis, and the pathobiology of cancer. This review explores the origins of intestinal organoids, their culturing environments, and their advancements in the realm of precision medicine. It also addresses the current challenges and outlines future prospects for development.
... Organoids mimic the functionality and organization of organ tissues and have become an invaluable tool for the in vitro study of biological processes, development, and diseases [13][14][15][16]. In the field of livestock biotechnology, organoid-based systems have been used for disease modeling, the determination of host pathogens interactions, and nutritional research to improve the productivity of livestock [17][18][19]. ...
Background
Deoxynivalenol (DON) is a mycotoxin that has received recognition worldwide because of its ability to cause growth delay, nutrient malabsorption, weight loss, emesis, and a reduction of feed intake in livestock. Since DON-contaminated feedstuff is absorbed in the gastrointestinal tract, we used chicken organoids to assess the DON-induced dysfunction of the small intestine.
Results
We established a culture system using chicken organoids and characterized the organoids at passages 1 and 10. We confirmed the mRNA expression levels of various cell markers in the organoids, such as KI67 , leucine-rich repeat containing G protein-coupled receptor 5 ( Lgr5 ), mucin 2 ( MUC2 ), chromogranin A ( CHGA ), cytokeratin 19 ( CK19 ), lysozyme ( LYZ ), and microtubule-associated doublecortin-like kinase 1 ( DCLK1 ), and compared the results to those of the small intestine. Our results showed that the organoids displayed functional similarities in permeability compared to the small intestine. DON damaged the tight junctions of the organoids, which resulted in increased permeability.
Conclusions
Our organoid culture displayed topological, genetic, and functional similarities with the small intestine cells. Based on these similarities, we confirmed that DON causes small intestine dysfunction. Chicken organoids offer a practical model for the research of harmful substances.
... In this cluster, we find a variety of genes (e.g. Reg3b, Reg3g, Agr2) indicating resolution and repair of the cellular environment (30,70,71), together with Mxra7, encoding Matrix remodelling associated 7 protein implicated in wound-healing (72). Additionally, RNA processing and translation-related genes (Pcbp2, Eef1b2, Eef1a1, Sf3b1, Rbm5) suggest heightened cellular activity (73)(74)(75). ...
The infective L3 larvae of Heligmosomoides polygyrus migrate to the small intestine where they take up residence in the submucosa, triggering the formation of complex granulomas around the parasite. Here, we employ spatial transcriptomics to elucidate the transcriptional intricacies and cell-cell interactions of the murine small intestine under both steady-state conditions and in response to H. polygyrus infection. Our findings unveil distinct transcriptional signatures in the crypt zone, villi, and granulomas, providing nuanced insights into the molecular dynamics of the host response to parasitic infection. Molecular characterization of H. polygyrus granulomas reveals unique cellular compositions within distinct clusters, shedding light on localized immune activation and cellular dynamics. Utilizing deconvolution techniques, we uncovered common and infection-specific signatures of cell type colocalization, and identified potential ligand-receptor pairs that may mediate communication between the granuloma tissue and the epithelial crypt cells. Additionally, our study highlights the upregulation of genes such as Ccl9, Fcer1g and Tmsb4x within granulomas, suggesting roles in type 2 inflammation, and genes (e.g Reg3b and Mxra7) associated with wound healing and tissue repair. These results not only enhance our understanding of the murine small intestine's transcriptional landscape but also provide a platform for exploring host-pathogen interactions. The comprehensive analysis presented here contributes to a holistic comprehension of tissue-specific responses during parasitic infections, offering valuable insights for targeted therapeutic interventions.
... Enhancement of the RSC induction offers beneficial effects on intestinal regeneration. A recently reported novel combination comprises eight components (8C) designed to mimic the authentic regenerative state of intestinal organoids (61). Compared to the original culture media, 8C led to a generation of hyper-proliferative organoids enriched for injury-associated regenerative phenotype. ...
Tissue-specific adult stem cells are pivotal in maintaining tissue homeostasis, especially in the rapidly renewing intestinal epithelium. At the heart of this process are leucine-rich repeat-containing G protein-coupled receptor 5-expressing crypt base columnar cells (CBCs) that differentiate into various intestinal epithelial cells. However, while these CBCs are vital for tissue turnover, they are vulnerable to cytotoxic agents. Recent advances indicate that alternative stem cell sources drive the epithelial regeneration post-injury. Techniques like lineage tracing and single-cell RNA sequencing, combined with in vitro organoid systems, highlight the remarkable cellular adaptability of the intestinal epithelium during repair. These regenerative responses are mediated by the reactivation of conserved stem cells, predominantly quiescent stem cells and revival stem cells. With focus on these cells, this review unpacks underlying mechanisms governing intestinal regeneration and explores their potential clinical applications.
... This complementation of the traditional media (Rspondin, EGF, and Noggin) facilitated faster and stronger growth and proliferation than before. In addition, the novel model exhibited a more complex crypt-villus structure and might thus be used in the future in intestinal regeneration research for the treatment of COVID-19associated intestinal symptoms [78]. Nikolaev et al. developed a tubular intestinal organoid to maintain longterm homeostasis. ...
The novel COVID-19 pneumonia caused by the SARS-CoV-2 virus poses a significant threat to human health. Scientists have made significant efforts to control this virus, consequently leading to the development of novel research methods. Traditional animal and 2D cell line models might not be suitable for large-scale applications in SARS-CoV-2 research owing to their limitations. As an emerging modelling method, organoids have been applied in the study of various diseases. Their advantages include their ability to closely mirror human physiology, ease of cultivation, low cost, and high reliability; thus, they are considered to be a suitable choice to further the research on SARS-CoV-2. During the course of various studies, SARS-CoV-2 was shown to infect a variety of organoid models, exhibiting changes similar to those observed in humans. This review summarises the various organoid models used in SARS-CoV-2 research, revealing the molecular mechanisms of viral infection and exploring the drug screening tests and vaccine research that have relied on organoid models, hence illustrating the role of organoids in remodelling SARS-CoV-2 research.
... Intestinal tissues were harvested after 2, 4, 6, 8, 10, and 24 h of BrdU injection. (C) Heatmap depicts transcript levels of fetal/regenerative marker genes and regenerative stem-cell-associated genes that are highly expressed at day 3 postirradiation (GSE165157, 16 RNA-seq, n = 2 biological replicates per time point). (D) GSEA confirms that gene signatures of regenerative epithelium, fetal spheroid, and revival stem cells 8,12,17 are elevated at day 3 post-irradiation (GSE165157, 16 crypt cells, n = 2 biological replicates per time course, Kolmogorov-Smirnov test, p < 0.001). ...
... (C) Heatmap depicts transcript levels of fetal/regenerative marker genes and regenerative stem-cell-associated genes that are highly expressed at day 3 postirradiation (GSE165157, 16 RNA-seq, n = 2 biological replicates per time point). (D) GSEA confirms that gene signatures of regenerative epithelium, fetal spheroid, and revival stem cells 8,12,17 are elevated at day 3 post-irradiation (GSE165157, 16 crypt cells, n = 2 biological replicates per time course, Kolmogorov-Smirnov test, p < 0.001). See expanded panel in Figure S1E. ...
... Organoid research involves multiple tissues and organs, including the brain, fat, liver, and intestine [28][29][30][31]. Early organoid studies focused on single organoids without the presence of other cellular components or other organoid components [5], and such studies lacked an accurate reproduction of the organoid microenvironment. ...
Intestinal organoids have emerged as powerful model systems for studying the complex structure and function of the intestine. However, there is a lack of widely applicable methods for the collection, labeling, and imaging of intestinal organoids. In this study, we developed a novel method for loading and labeling intestinal organoids, a method that efficiently collects the organoids and facilitates imaging of their three-dimensional (3D) structure. Based on this strainer platform, mouse intestinal organoids were adequately collected and immobilized, facilitating the immunolabeling workflow to target proteins of the organoids. After evaluation, the strainer size of 40 μm was considered to be more conducive to the collection and labeling of mouse intestinal organoids. More extensive research on organoids of multiple types and species origins will contribute to broadening the applicability of the methodology. Overall, our study proposes an innovative workflow for loading and analyzing intestinal organoids. The combination of a strainer-based collection method, fluorescent labeling, and 3D reconstruction provides valuable insights into the organization and complexity of these tissue models, thereby offering new avenues for investigating intestinal development, disease modeling, and drug discovery.
