Bulk RNA-seq of MilliporeSigma SCC300 human iPSC-derived colon organoids compared with undifferentiated human iPSCs and MilliporeSigma SCC321 human patient-derived colon organoids
Ontology highlight
ABSTRACT: Experiment intended to obtain expression profiles of iPSC-derived human colon organoids compared to undifferentiated human iPSCs and a patient-derived colon organoid line
Project description:Transcriptomic profiles of 6 commercially-available human patient-derived gastrointestinal organoid lines were obtained and compared to transcriptomic profile of a commercially available human iPSC-induced colon organoid line. Transcriptomic profile of iPSC-derived human colon organoid line was compared after culture in either Corning growth-factor-reduced Matrigel (Corning 356231) or MilliporeSigma growth-factor-reduced ECMGel (E6909)
Project description:Human colon organoids were maintained in culture medium alone or exposed to lipopolysaccharide with a combination of three pro-inflammatory cytokines (tumor necrosis factor-a, interleukin-1b and interferon-g [LPS-cytokines]) to mimic the environment in the inflamed colon. Untreated organoids and those exposed to LPS-cytokines were concomitantly treated with a multi-mineral product that has previously been shown to improve barrier structure/function. The organoids were subjected to proteomic analysis to obtain a broad view of the protein changes induced by these interventions. In parallel, confocal fluorescence microscopy and trans-epithelial electrical resistance measurements were used to assess barrier structure/function. The LPS-cytokines altered expression of multiple proteins that influence innate immunity and promote inflammation. Most of these were unaffected by the multi-mineral intervention, though a subset of inflammation-related proteins including fibrinogen-b and -g chains, phospholipase A2 and SPARC was down-regulated in the presence of the multi-mineral intervention; another subset of proteins with anti-inflammatory, antioxidant or anti-microbial activity was up-regulated by multi-mineral treatment. When used alone, the multi-mineral intervention strongly up-regulated proteins that contribute to barrier formation and tissue strength. Concomitant treatment with LPS-cytokines did not inhibit barrier formation in response to the multi-mineral intervention.
Project description:Background and aims: Cell-cell adhesion structures (desmosomes and, especially, tight junctions) are known to play important roles in control of transepithelial permeability in the colon. The involvement of cell-matrix interactions in permeability control is less clear. The goals of the present study were to: i) determine if disruption of colon epithelial cell interactions with the extracellular matrix alters permeability control and ii) determine if increasing the elaboration of protein components of cell-matrix adhesion complexes improves permeability control and mitigates the effects of cell-matrix disruption. Results: Treatment of colon organoids with Aquamin® increased the expression of multiple basement membrane and hemidesmosomal proteins as well as keratin 8 and 18. TEER values were higher in the presence of Aquamin® than they were under control conditions. Conclusions: These findings provide evidence that cell-matrix interactions contribute to permeability control in the colon. They suggest that the elaboration of proteins important to cell-matrix interactions can be increased in human colon organoids by exposure to a multi-mineral natural product. Increasing the elaboration of such proteins may help to mitigate the consequences of disrupting cell-matrix interactions on permeability control.
Project description:Hepatic organoids are a recent innovation in in vitro modelling. Initial studies suggest organoids better recapitulate the liver phenotype in vitro compared to pre-existing proliferative cell models. However, their propensity for drug metabolism and detoxification remains poorly characterised. A global proteomic profiling of undifferentiated and differentiated hepatic murine organoids and donor-matched livers was therefore performed to assess both their similarity to liver tissue and DMET expression. iTRAQ analysis revealed 4,405 proteins commonly detected across all sample types. Differentiation of organoids significantly increased the expression of multiple CYP450s, phase II enzymes, liver biomarkers and hepatic transporters. While the final phenotype of differentiated organoids is distinct from liver tissue, they contain multiple DMET proteins necessary for liver function and drug metabolism, such as CYP450 3A, GSTA and MDR1A. Hepatic organoids may therefore represent an attractive novel model for hepatotoxicity testing, although further experimentation, optimisation and characterisation is needed relative to pre-existing models to fully contextualise their use as a putative in vitro model of DILI.
Project description:We aimed to investigate genome wide DNA methylation changes in human colon organoids in which the BRAF-V600E mutation was introduced by means of CRISPR genome editing and in BRAF-wildtype human colon cancer organoids. DNA methylation analysis was performed using the Infinium Methylation EPIC beadchip at the Pathology department of the UMC Utrecht (The Netherlands).
Project description:Background & Aims: We have recently established long-term culture conditions under which single crypts or stem cells derived from murine small intestine expand over long periods of time. Growing crypts undergo multiple crypt fission events, whilst simultaneously generating villus-like epithelial domains in which all differentiated cell types are present. We have now adapted the culture conditions to grow similar epithelial organoids from mouse colon and human small intestine and colon. Methods: Based on the murine small intestinal culture system, we optimized the murine and human colon culture system. Results: Addition of Wnt3A to the growth factor cocktail allowed mouse colon crypts to expand indefinitely. Further addition of nicotinamide, a small molecule Alk inhibitor and a p38 inhibitor was essential for long-term human small intestine and colon culture. The culture system also allowed growth of murine Apcmin adenomas, human colorectal cancer and human esophageal metaplastic Barrett’s epithelium. Conclusion: The culture technology should be widely applicable as a research tool for infectious, inflammatory and neoplastic pathologies of the human gastrointestinal tract. Moreover, regenerative applications may become feasible with ex vivo expanded intestinal epithelia. Human organoids were grown embedded in Matrigel in HISC (Human intestinal stem cell culture) medium. Additionally, human small intestinal crypts and villi were isolated independently from a freshly operated sample. RNA was isolated using the RNeasy Micro kit (Qiagen). Samples were labled according to Agilent guidelines with Cy3, whereas human reference RNA (Stratagene) was labeled in Cy5. Feature Extraction Software was used to extract and normalize data.
Project description:Molecular characterization of tissue-resident memory T cells cultured with or without donor-matched adult stem cell-derived intestinal organoids. Blood derived immune cells were also isolated and cultivated with autologous intestinal organoids for comparison and characterization. All conditions were derived from 3 human individuals and all samples were sequenced after 24h of in vitro culture. Data provides insights on circulating and tissue-resident immune cell populations, how these differentially interact with the epithelium and how these interactions shape both immune and epithelial cell states.