Whole-genome RNAsequencing of intestinal organoids and tumoroids
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE153160 | GEO | 2022/05/09
REPOSITORIES: GEO
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Project description:Whole-genome RNAsequencing of intestinal organoids and tumoroids
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Project description:We isolated and cultured Intestinal Organoids from Mouse. To trace the metabolism of fructose, intestinal organoids were cultured in unlabeled fructose-containing DMEM/F12 medium (10 mM D-fructose, 0 mM D-glucose, 2.5 mM L-glutamine) for 12 hr, and then switched into 13C labeled fructose-containing medium (10 mM U-[13C] D-fructose, Sigma-aldrich) for 6 hr.
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2022-12-06 | GSE219127 | GEO
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2020-12-09 | ST001621 | MetabolomicsWorkbench
Project description:Intestinal ischemia-reperfusion (IR) injury is associated with high mortality rates, which have not improved in the past decades despite advanced insight in its pathophysiology using in vivo animal and human models. The inability to translate previous findings to effective therapies emphasizes the need for a physiologically relevant in vitro model to thoroughly investigate mechanisms of IR-induced epithelial injury and test potential therapies. In this study, we demonstrate the use of human small intestinal organoids to model IR injury by exposing organoids to hypoxia and reoxygenation (HR). A mass-spectrometry-based proteomics approach was applied to characterize organoid differentiation and decipher protein dynamics and molecular mechanisms of IR injury in crypt-like and villus-like human intestinal organoids.
2021-01-11 | PXD022999 | Pride
Project description:Cervical cancer is a common gynecological malignancy, often caused by high-risk human papillomavirus. There is a paucity of human-derived culture systems to study cervical epithelium and the cancers derived thereof. Here, we describe a long-term culturing protocol for ecto- and endocervical epithelia, which generates 3D organoids that stably recapitulate the two tissues of origin. In the example of successful HSV-1 infection, we show that the organoid-based cervical models serve as promising platforms for studying sexually transmitted pathogens. Starting from Pap-brush material, a small biobank of patient-derived tumoroids was established that retained the causative HPV genomes. One of these uniquely carried the poorly characterized HPV30 subtype, implying a potential role in carcinogenesis. The tumoroids displayed differential responses to common chemotherapeutics and grew as xenografts in mice. This study describes an experimental platform for cervical (cancer) research and for future personalized medicine approaches. Purpose: Transcriptome analysis was performed on organoids/tissues derived from human ecto- and endocervix as well as associated malignancies to characterize the new organoid-based platform. Methods: Ectocervical organoids and tumoroid lines were cultured in M7 media, whereas endocervical organoids in M7+4 media prior harvesting for RNA extraction (see paper for details). Additionally, RNA was extracted from healthy ecto-and endocervical tissue biopsies as well as from primary cells collected via Pap brush method from cervical cancer patients. The libraries were prepared by the Utrecht Sequencing Facility (USEQ) based on polyA enrichment. Sequencing was performed on an Illumina NextSeq500 by using 75-bp single-end or paired-end sequencing. Reads were aligned to the human reference genome (GRCh37) using Burrows-Wheeler Aligner (BWA) (v0.5.9). Viral sequences of different HPV subtypes were detected in the RNA-seq data of the different tumoroid lines and tissues by using VirusSeq-CLI, a wrapper around the VirusSeq pipeline. Results: Ectocervical and endocervical organoids display distinct transcriptomic profiles that can be maintained in cultures for long term. Similarily, the the gene expression profile of tumor organoids clusters apart from healthy counterparts and shows evident tumor-like characteristics. Tumor organoids show expression of causative HPV oncogenes.
2021-03-05 | GSE168244 | GEO
Project description:The gastrointestinal microbiota is involved in the development of various diseases, such as obesity and diabetes, by affecting nutrient acquisition, energy balance, and metabolic signaling of the host. However, the underlying mechanisms of these host-microbiota interactions remain unclear. The aim of this study was to characterize effects of the microbiota on the host epithelium using a novel gastrointestinal model based on mouse organoids. We have explored the transcriptional response of organoids upon exposure to short chain fatty acids (SCFA) and products generated by two conspicuous members of the gastrointestinal microbiota; Akkermansia muciniphila and Faecalibacterium prausnitzii. We observed that A. muciniphila metabolites affect a large variety of transcription factors and genes involved in cellular lipid metabolism and growth, supporting previous in vivo findings. F. prausnitzii products exerted only a weak effect on the host transcription profile. While, A. muciniphila, and its main metabolite propionate, both stimulate fasting-induced adipose factor/angiopoietin-like protein 4 (Fiaf/Angptl4) and Ppar? expression, important regulators of lipolysis and satiety. This work illustrates that specific members of the microbiota and their metabolites differentially modulate epithelial transcription in mouse organoid lines. Organoid culture: In short, murine (WT C57BL/6) small intestinal crypts were isolated and embedded in 250 µl Matrigel (BD Biosciences) and submerged in 500 ul basal culture medium supplemented with penicillin/streptomycin, HEPES, Glutamax, N2, B27, N-acetylcysteine, and the murine growth factors EGF, noggin, and R-spondin-1 (ENR), as previously described (Sato et al., 2009. Nature 459:262–5). Organoids were passaged every 7 days with a 1:4 splitting ratio. Exposure to bacterial cultures and SCFA: A. muciniphila (ATTC BAA-835) was grown anaerobically at 37C in a basal liquid medium which contained (per liter of deionized water) the following: 0.4g KH2PO4, 0.669g, Na2HPO4 + 2H2O, 0.3g NH4CL, 0.3gNaCl, 0.1g MgCl2 + 6H2O, 10g casitone, 1mM L-threonine, 1 ml trace mineral solution, 5mM L-fucose, and 5mM D-glucose. F. prausnitzii strain A2-165 (DSM 17677) was grown anaerobically at 37ºC a liquid medium which contained (per liter of deionized water) the following: 10 g casitone, 2.5 g yeast extract, 4 g NaHCO3, 1 ml resazurine (0.1%w/v), 0.45g K2HPO4, 0.45g KH2PO4, 0.9g (NH4)2SO4, 0.9g NaCL, 0.09g MgSO4, 0.09g CaCL2, 2.5 mM acetate, 9 mM propionate, 1 mM n-valerate, 1 mM isovalerate, 1 mM isobutyrate, 0.28g KOH, 2.5 ml ethanol (95%), 10 mg haemin,, 10 ug biotin, 10 ug cobalamin, 30 ug para-aminobenzoic acid, 50 ug folic acid, 150 ug pyridoxamine, 1 g L-cysteine, and 25 mM glucose (Duncan et al., 2002. Int. J. Syst. Evol. Microbiol. 52:2141–6). The concentration of the following organic acids and sugars were determined in the culture medium before and after growth using a high performance liquid chromatography (HPLC) equipped with a Shodex Sugar SH-G and Shodex SH1821 column as has been described previously (Stams et al., 1993. Appl. Environ. Microbiol. 59:1114–9): glucose, mannose, galactose, L-fucose, glucose-N-acetylglucosamine, lactate, formate acetate, propionate, 1,2-propendiol and butyrate. Overnight-grown cultures were pelleted by centrifugation at 20 000 ×g for 10 min and supernatant was collected. At t=7 days after splitting organoids were exposed to 250 μl A. muciniphila supernatant, 250 μl unconditioned A. muciniphila culture medium, 85 μl F. prausnitzii supernatant, or 85 μl F. prausnitzii culture medium for 3 hours at 37ºC, prior to RNA extraction. 250 μl A. muciniphila supernatant and culture medium Short chain fatty acids (SCFA) sodium butyrate, sodium propionate, and sodium acetate (Sigma-Aldrich, Zwijndrecht, the Netherlands) were administered at a final concentration of 5 mM. At t=7 days after splitting mature organoids were exposed to individual SCFAs for 3 hours at 37ºC, prior to RNA extraction. Data is presented from n = 4 independent biological replicates from one line of organoids. total RNA was isolated from mouse intestinal organoids exposed for 3h to SCFA and strain-specific culture medium: Am- (organoids exposed to A. muciniphila culturing medium), Am+ (A. muciniphila-conditioned medium), Fp- (F. prausnitzii-culturing medium), Fp+ (F. prausnitzii-conditioned medium), Ace (acetate), But (butyrate), Pro (propionate) and Con (standard organoid culturing medium).
2014-07-23 | E-GEOD-59644 | biostudies-arrayexpress
Project description:The thyroid maintains systemic homeostasis by regulating serum thyroid hormone concentrations. Here we report the establishment of adult stem cell-derived three-dimensional (3D) organoids representing murine and human thyroid follicular cells (TFCs). The TFC organoids (TFCO) harbour the complete machinery of hormone production as visualised by the presence of colloid in the lumen and by the presence of essential transporters and enzymes in the polarised epithelial cells that surround a central lumen. Both the established murine as human thyroid organoids express canonical thyroid markers PAX8 and NKX2.1, while the thyroid hormone precursor thyroglobulin is expressed at comparable levels to tissue. Single cell RNA sequencing and transmission electron microscopy confirm that TFCOs phenocopy primary thyroid tissue. Thyroid hormones are readily detectable in conditioned medium of human TFCOs.
2021-12-06 | GSE183961 | GEO
Project description:The thyroid maintains systemic homeostasis by regulating serum thyroid hormone concentrations. Here we report the establishment of adult stem cell-derived three-dimensional (3D) organoids representing murine and human thyroid follicular cells (TFCs). The TFC organoids (TFCO) harbour the complete machinery of hormone production as visualised by the presence of colloid in the lumen and by the presence of essential transporters and enzymes in the polarised epithelial cells that surround a central lumen. Both the established murine as human thyroid organoids express canonical thyroid markers PAX8 and NKX2.1, while the thyroid hormone precursor thyroglobulin is expressed at comparable levels to tissue. Single cell RNA sequencing and transmission electron microscopy confirm that TFCOs phenocopy primary thyroid tissue. Thyroid hormones are readily detectable in conditioned medium of human TFCOs.
2021-12-06 | GSE183962 | GEO