Physiological hypoxia improves growth and functional differentiation of human intestinal epithelial organoids.
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA899901 | ENA |
REPOSITORIES: ENA
Similar Datasets
Project description:Physiological hypoxia improves growth and functional differentiation of human intestinal epithelial organoids [scRNA-seq]
| PRJNA904706 | ENA
Project description:The intestinal epithelium is an immunologically active barrier adapted for a low oxygen environment. Its role is implicated in the pathophysiology of various diseases, including inflammatory bowel disease (IBD) and colorectal cancer. Patient-derived intestinal epithelial organoids (IEOs) mimic the architecture and cell type composition of the intestine and can be used for disease modeling and personalized drug screening. IEOs are usually cultured in atmospheric oxygen concentrations (20% oxygen) without accounting for the physiological hypoxia in the colonic epithelium, where the oxygen concentration range from 3% to <1%. Previously, we showed that human colon derived IEOs (colonoids) are viable and respond to the pro-inflammatory cytokines TNF and IL17 after short-term (40 hours) cultivation in low (2%) oxygen. We suggested that recapitulating the in vivo physiological oxygen environment (i.e., physioxia) will enhance the translational value of intestinal organoids as pre-clinical models. Here we evaluate whether human colonoids can be established and cultured in physioxia for the entire in vitro culture period and compare growth, differentiation, and immunological responses in colonoids at 2% and 20% oxygen. Growth from single cells to differentiated organoids was monitored by brightfield images captured throughout cultivation and evaluated with a linear mixed model. Cell composition in undifferentiated vs. differentiated colonoids was identified by immunofluorescence staining of cell markers and single-cell RNA-sequencing. Enrichment analysis was used to identify transcriptomic differences within cell populations. Pro-inflammatory stimuli induced chemokines and Neutrophil gelatinase-associated lipocalin (NGAL) release were analyzed by Multiplex profiling and ELISA. Direct response to a lower oxygen level was analyzed by enrichment analysis of bulk RNA sequencing data. Colonoids established in a 2% oxygen environment acquired a significantly larger cell mass compared to a 20% oxygen environment. There were significantly more cells with proliferation potential (KI67 positive) in undifferentiated than in differentiated colonoids; and significantly higher expression of cell markers for goblet cells (MUC2) and absorptive cells (MUC2 negative, CK20 positive) in differentiated colonoids. Enteroendocrine cells (CGA positive) were only present in differentiated colonoids. No differences in expression of any of the cell marker proteins were found between colonoids cultured in 2% and 20% oxygen, but the single-cell RNA-sequencing analysis identified differences in the transcriptome within stem-, progenitor- and differentiated cell clusters. Both colonoids grown at 2% and 20% oxygen secreted CX3CL1, CXCL2, CXCL5, CXCL6, CXCL10, CXCL12, CCL20, CCL25, and NGAL upon TNF + poly(I:C) treatment. However, there appeared to be a lower pro-inflammatory response in 2% oxygen. Reducing the oxygen environment from 20% to 2% in differentiated colonoids altered the expression of genes related to differentiation, metabolism, mucus lining, and immune networks. Our results suggest that colonoids studies can and should be performed in physioxia when the resemblance to in vivo conditions is important.
2022-12-02 | GSE217663 | GEO
Project description:The intestinal epithelium is an immunologically active barrier adapted for a low oxygen environment. Its role is implicated in the pathophysiology of various diseases, including inflammatory bowel disease (IBD) and colorectal cancer. Patient-derived intestinal epithelial organoids (IEOs) mimic the architecture and cell type composition of the intestine and can be used for disease modeling and personalized drug screening. IEOs are usually cultured in atmospheric oxygen concentrations (20% oxygen) without accounting for the physiological hypoxia in the colonic epithelium, where the oxygen concentration range from 3% to <1%. Previously, we showed that human colon derived IEOs (colonoids) are viable and respond to the pro-inflammatory cytokines TNF and IL17 after short-term (40 hours) cultivation in low (2%) oxygen. We suggested that recapitulating the in vivo physiological oxygen environment (i.e., physioxia) will enhance the translational value of intestinal organoids as pre-clinical models. Here we evaluate whether human colonoids can be established and cultured in physioxia for the entire in vitro culture period and compare growth, differentiation, and immunological responses in colonoids at 2% and 20% oxygen. Growth from single cells to differentiated organoids was monitored by brightfield images captured throughout cultivation and evaluated with a linear mixed model. Cell composition in undifferentiated vs. differentiated colonoids was identified by immunofluorescence staining of cell markers and single-cell RNA-sequencing. Enrichment analysis was used to identify transcriptomic differences within cell populations. Pro-inflammatory stimuli induced chemokines and Neutrophil gelatinase-associated lipocalin (NGAL) release were analyzed by Multiplex profiling and ELISA. Direct response to a lower oxygen level was analyzed by enrichment analysis of bulk RNA sequencing data. Colonoids established in a 2% oxygen environment acquired a significantly larger cell mass compared to a 20% oxygen environment. There were significantly more cells with proliferation potential (KI67 positive) in undifferentiated than in differentiated colonoids; and significantly higher expression of cell markers for goblet cells (MUC2) and absorptive cells (MUC2 negative, CK20 positive) in differentiated colonoids. Enteroendocrine cells (CGA positive) were only present in differentiated colonoids. No differences in expression of any of the cell marker proteins were found between colonoids cultured in 2% and 20% oxygen, but the single-cell RNA-sequencing analysis identified differences in the transcriptome within stem-, progenitor- and differentiated cell clusters. Both colonoids grown at 2% and 20% oxygen secreted CX3CL1, CXCL2, CXCL5, CXCL6, CXCL10, CXCL12, CCL20, CCL25, and NGAL upon TNF + poly(I:C) treatment. However, there appeared to be a lower pro-inflammatory response in 2% oxygen. Reducing the oxygen environment from 20% to 2% in differentiated colonoids altered the expression of genes related to differentiation, metabolism, mucus lining, and immune networks. Our results suggest that colonoids studies can and should be performed in physioxia when the resemblance to in vivo conditions is important.
2022-12-02 | GSE218623 | GEO
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:Retinoic acid promotes the in vitro growth, patterning and improves the cellular composition of human pluripotent stem-cell-derived intestinal organoids
2022-09-02 | GSE212333 | GEO
Project description:Metabolic Maturation Media Improves Physiological Function of Human iPSC-derived Cardiomyocytes
| PRJNA635378 | ENA
Project description:Primary tissue-derived epithelial organoids are a physiologically relevant in vitro intestinal model that have been implemented for both basic research and drug development applications. The existing method of culturing intestinal organoids in surface-attached native extracellular matrix (ECM) hydrogel domes is not readily amenable to large-scale culture and contributes to culture heterogeneity. We have developed a method of culturing intestinal organoids within suspended basement membrane extract (BME) hydrogels of various geometries, which streamlines the protocol, increases the scalability, enables kinetic sampling, and improves culture uniformity without specialized equipment or additional expertise. We demonstrate the compatibility of this method with multiple culture formats, and provide examples of suspended BME hydrogel organoids in downstream applications: implementation in a medium-throughput drug screen and generation of Transwell monolayers for barrier evaluation. The suspended BME hydrogel culture method will allow intestinal organoids, and potentially other organoid types, to be used more widely and at higher throughputs than previously possible.
2023-09-11 | GSE226539 | GEO
Project description:To study the responses to physiological and pathological hypoxia, we performed gene expression profiling analysis of RNA-seq data for murince corneal epithelial cells preconditioned under 3 oxygen levels.
2023-11-02 | GSE218718 | GEO
Project description:Circadian rhythms are 24h changes in biochemical, physiological and behavioural processes. When ciricadian rhythm are perturbed, like in shiftwork, lead to negitive health outcomes, such as cancer. We assess circadian transcriptional regulation in healthy (APC+_+) and tumourigenic (APCmin) organoids with (BMAL1_WT) or without (BMAL1_Mut) a functional circadain clock. Our results show that the clock controls several genes involved in stem cell signalling pathways.
2021-10-04 | GSE157357 | GEO
Project description:Physiological shear stress, produced by blood flow, homeostatically regulates the phenotype of pulmonary endothelial cells exerting anti-inflammatory and anti-thrombotic actions and maintaining normal barrier function. In the pulmonary circulation hypoxia, due to high altitude or diseases such as COPD, causes vasoconstriction, increased vascular resistance and pulmonary hypertension. Hypoxia-induced changes in endothelial function play a central role in the development of this pulmonary hypertension. However, the direct interactive effects of hypoxia and shear stress on the pulmonary endothelial phenotype have not been extensively studied. We cultured human pulmonary microvascular endothelial cells (HPMEC) in normoxia or hypoxia while subjected to physiological shear stress or in static conditions. Unbiased proteomics was used to identify hypoxia-induced changes in protein expression. Using publicly available single cell RNA-seq datasets, differences in gene expression between the alveolar endothelial cells from COPD and healthy lungs were identified. 60 proteins were identified in HPMEC lysates whose expression changed in response to hypoxia in sheared but not in static conditions. mRNA for five of these (ERG, MCRIP1, EIF4A2, HSP90AA1 and DNAJA1) showed similar changes in the endothelial cells of COPD compared to healthy lungs. These data show that the proteomic responses of the pulmonary microvascular endothelium to hypoxia are significantly altered by shear stress and suggest that these differences are important in the development of hypoxic pulmonary vascular disease.
2023-02-17 | PXD036260 | Pride