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Culture associated DNA methylation changes impact on cellular function of Human Intestinal Organoids (Expression)

ABSTRACT: Culture associated DNA methylation changes impact on cellular function of Human Intestinal Organoids (Expression)

PROVIDER: PRJEB51685 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	ERR9286880.fastq.gz	Fastqsanger.gz
	ERR9286890.fastq.gz	Fastqsanger.gz
	ERR9286900.fastq.gz	Fastqsanger.gz
	ERR9286910.fastq.gz	Fastqsanger.gz
	ERR9286871.fastq.gz	Fastqsanger.gz

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Culture associated DNA methylation changes impact on cellular function of Human Intestinal Organoids (Expression)

Project description:Human intestinal epithelial organoid models are rapidly emerging as novel experimental tools to investigate intestinal epithelial biology. A necessary aspect of organoid use is the passaging of cells and long term maintenance in culture. DNA methylation has been demonstrated to play a key role in regulating gene expression and cellular function. Here we explore the effect of culture duration, proinflammatory cytokine stimulation and differentiation on organoid DNA methylation. The experiment consists of RNA-seq of intestinal organoid cultures from paediatric ileum and colon.

2022-08-31 | E-MTAB-11548 | biostudies-arrayexpress

Culture associated DNA methylation changes impact on cellular function of Human Intestinal Organoids (Methylation)

Project description:Human intestinal epithelial organoid models are rapidly emerging as novel experimental tools to investigate intestinal epithelial biology. A necessary aspect of organoid use is the passaging of cells and long term maintenance in culture. DNA methylation has been demonstrated to play a key role in regulating gene expression and cellular function. Here we explore the effect of culture duration, proinflammatory cytokine stimulation and differentiation on organoid DNA methylation. The experiment consists of genome-wide DNA methylation profiling by Infinium HumanMethylationEPIC BeadChip of intestinal organoid cultures from paediatric ileum and colon. Our genome wide DNA methylation datasets represent a unique resource, which can be used by other researchers to validate their model systems.

2022-08-31 | E-MTAB-11545 | biostudies-arrayexpress

Culture-Associated DNA Methylation Changes Impact on Cellular Function of Human Intestinal Organoids.

Project description:Background & aimsHuman intestinal epithelial organoids (IEOs) are a powerful tool to model major aspects of intestinal development, health, and diseases because patient-derived cultures retain many features found in vivo. A necessary aspect of the organoid model is the requirement to expand cultures in vitro through several rounds of passaging. This is of concern because the passaging of cells has been shown to affect cell morphology, ploidy, and function.MethodsHere, we analyzed 173 human IEO lines derived from the small and large bowel and examined the effect of culture duration on DNA methylation (DNAm). Furthermore, we tested the potential impact of DNAm changes on gene expression and cellular function.ResultsOur analyses show a reproducible effect of culture duration on DNAm in a large discovery cohort as well as 2 publicly available validation cohorts generated in different laboratories. Although methylation changes were seen in only approximately 8% of tested cytosine-phosphate-guanine dinucleotides (CpGs) and global cellular function remained stable, a subset of methylation changes correlated with altered gene expression at baseline as well as in response to inflammatory cytokine exposure and withdrawal of Wnt agonists. Importantly, epigenetic changes were found to be enriched in genomic regions associated with colonic cancer and distant to the site of replication, indicating similarities to malignant transformation.ConclusionsOur study shows distinct culture-associated epigenetic changes in mucosa-derived human IEOs, some of which appear to impact gene transcriptomic and cellular function. These findings highlight the need for future studies in this area and the importance of considering passage number as a potentially confounding factor.

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Suspended hydrogel culture as a method to scale up intestinal organoids

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

scRNA-seq of pluripotent stem cell derived human intestinal organoids grown in suspension culture

Project description:Here, we used single-cell RNA-sequencing (scRNA-seq) to profile pluripotent stem cell derived human intestinal organoids (HIOs) grown in suspension culture after 28 days of in vitro growth. Grown in minigut media supplemented with EGF.

2022-02-04 | E-MTAB-11347 | biostudies-arrayexpress

Quantitative mass spectrometry analysis of intestinal organoids

Project description:Organoids have the potential to bridge 3D cell culture to tissue physiology by providing a model resembling in vivo organs. We propose here a SILAC method to measure protein expression changes in intestinal organoids under different experimental conditions. With the combined use of quantitative mass spectrometry, SILAC and organoid culture, we validated the approach and showed that large-scale proteome variations can be measured in an “organ-like” system under variable conditions.

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RNA-seq of wildtype mouse small intestinal organoids grown in Matrigel or in/on collagen.

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2021-04-01 | E-MTAB-10082 | biostudies-arrayexpress

An Integrative Transcriptome – Proteome Approach Reveals a Robust Expression Pattern of Small Intestinal Epithelial Organoids from Germ-Free and Microbiota Associated Donors

Project description:Intestinal epithelial organoids established from resected gut tissue have become a widely used research tool. However, it remains unclear how environmental cues and other sources of variation before, during and after establishment impact on organoid properties. Divergent microbiota composition in separately held mouse lines has been identified as a confounding factor in murine in vivo studies. To probe the effect of donor microbiota on organoids, we analyzed the proteomes and transcriptomes of primary organoid cultures established from colonized and germ-free mice, and further compared them to their tissue of origin and to commonly used cell lines. The long-term global impact of donor microbiota on organoid expression patterns was negligible. Instead, stochastic culture-to-culture differences accounted for a moderate variability between independently established organoids. Integration of transcriptome and proteome datasets revealed an organoid-typic expression signature comprising 14 transcripts and 10 proteins that distinguished organoids across all donors from murine epithelial cell lines and fibroblasts. This signature, which closely mimicked expression patterns in the gut epithelium, included high levels of the inflammasome scaffold protein ASC in organoids which was lacking in commonly used cell lines. Mining of the transcriptome dataset uncovered similar high expression of also other inflammasome components, including Naip1-6, Nlrc4, and Caspase-1 in all organoids compared to the reference cell lines. Taken together, these results reveal a robust global expression pattern in organoids established from colonized and germ-free animals and suggest that organoids represent a more suitable culture model than immortalized cell lines for studies of intestinal epithelial inflammasomes.

2020-03-05 | PXD016339 | Pride

The Culture of Advanced/Recurrent/Metastatic Colorectal Cancer Organoids and Drug Screening

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Limited oxygen availability in standard cell culture alters metabolism and function in terminally differentiated cells

Project description:<p>Cell culture is generally considered to be hyperoxic. However, the importance of cellular oxygen consumption is often underappreciated, with rates of oxygen consumption often sufficient to cause hypoxia at cell monolayers. We initially focused on cultured adipocytes as a terminally differentiated cell-type with substantial oxygen consumption rates to support diverse cellular functions. Under standard conditions, cultured adipocytes are hypoxic and highly glycolytic. Increasing oxygen diverted glucose flux toward mitochondria and resulted in thousands of gene expression changes that pointed toward alleviated physiological transcriptional responses to hypoxia. Phenotypically, providing more oxygen increased adipokine secretion and rendered adipocytes more sensitive to insulin and lipolytic stimuli. The functional benefits of increasing pericellular oxygen were transferable to other cellular systems including hPSC-derived hepatocytes and cardiac organoids. Our findings suggest that oxygen is limiting in many terminally-differentiated cell culture systems, and that controlling oxygen availability can improve the quality and translatability of cell models.</p>

2024-02-21 | MTBLS6677 | MetaboLights

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