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Deep Visual Proteomics advances human colon organoid models by revealing a switch to an in vivo-like phenotype upon xenotransplantation

ABSTRACT: Intestinal epithelial damage predisposes to chronic disorders like inflammatory bowel disease. The organoid model allows the cultivation, expansion and analysis of primary intestinal epithelial cells and has been instrumental in studying epithelial behavior in homeostasis and disease. Recent advances in organoid transplantation lay the base for studies of human epithelial cell behavior within the intestinal tissue context. It remained unclear how organoid transplantation into the colon affects epithelial phenotypes, which is key to assessing the model’s suitability to study human epithelial cells. We employed Deep Visual Proteomics, integrating AI-guided cell classification, laser microdissection, and an improved proteomics pipeline to study the human colon mucosa. Thereby, we created the first cell type-resolved proteomics dataset of human intestinal epithelial cells within human tissue, in vitro organoids, and the murine colon post-xenotransplantation. Our findings reveal that in vitro conditions induce a proliferative organoid phenotype, which is reversible upon transplantation and adjustment of organoid culturing conditions.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell, Colon

SUBMITTER: Mario Oroshi

LAB HEAD: Matthias Mann

PROVIDER: PXD052341 | Pride | 2025-09-11

REPOSITORIES: Pride

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			Action	DRS
	DVP_data.pg_matrix.tsv	Tabular
	DVP_data.pr_matrix.tsv	Tabular
	Post_et_al_organoid_DVP_metadata.tsv	Tabular
	bulk_data.pg_matrix.tsv	Tabular
	bulk_data.pr_matrix.tsv	Tabular

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Project description:Human intestinal epithelial organoids (IEO) culture models are rapidly emerging as novel experimental tools to investigate fundamental aspects of intestinal epithelial (patho)physiology. Cellular source and culture protocols vary between different IEO models and reliable markers for their characterization/validation are currently limited. DNA methylation has been demonstrated to play a key role in regulating gene expression and cellular function. This epigenetic mark is comparably stable and has been shown to reflect cellular identity such as tissue origin, developmental stage and age. Our genome wide DNA methylation datasets of purified human epithelium represent a unique resource, which can be used by other researchers to validate their model systems We provide the following datasets of genome-wide DNA methylation profiling by Infinium HumanMethylation450 BeadChip: Purified intestinal epithelial cells (EpCAM+) from paediatric ileum and colon, Non-epithelial mucosal cells (EpCAM-), Whole colonic mucosal biopsies, Intestinal organoid cultures from paediatric ileum and colon, Purified intestinal epithelial cells (EpCAM+) from foetal small intestine and foetal large intestine, Intestinal organoid cultures from foetal small intestine and foetal large intestine, Intestinal organoid cultures derived from induced pluripotent stem cells. Note: Some samples in this data set were previously submitted to ArrayExpress under accession number E-MTAB-3709. They're clearly marked in the âDescriptionâ field in sample annotations. Information about batch effect during sample handling is included in the experimental variable âblockâ. Batch effect is not massive but present, so it is recommended that batch correction is carried out in data analysis. Complementary RNA-seq data on the purified cells and organoids can be found in ArrayExpress at E-MTAB-5015 ( https://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-5015/ ).

Project description:Genetically engineered human pluripotent stem cells (hPSCs) have been proposed as a source for transplantation therapies and are rapidly becoming valuable tools for human disease modeling. However, many of the potential applications are still limited by the lack of robust differentiation paradigms that allow for the isolation of defined functional tissues. These challenges could be overcome by the use of adult tissue stem cells derived from hPSCs, as their restricted potential could limit the differentiation towards other undesired linages, and allow in vitro expansion and long- term propagation of fully differentiated tissue. To isolate adult stem cells from hPSCs, we applied genome-editing to generate an LGR5-GFP reporter system and subsequently developed a differentiation protocol for human intestinal tissue comprising an adult stem cell niche and all major cell types of the adult intestine. This novel derivation protocol is highly robust and even permits the isolation of intestinal organoids without the LGR5 reporter. Transcriptional profiling, electron microscopy and functional analysis revealed that such human organoid cultures could be derived with high purity, and a composition and morphology similar to that of cultures obtained from human biopsies. Importantly, hPSC-derived organoids responded to the canonical signaling pathways that control self-renewal and differentiation in the adult human intestinal stem cell compartment. With our ability to genetically engineer hPSCs using site-specific nucleases, this adult stem cell system provides a novel platform by which to study human intestinal disease in vitro. RNA from primary organoid samples was isolated from organoid lines that were both cultured for 1-6 months and derived from duodenum, ileum, or rectum biopsies of human subjects as described previously (Sato et al., Gastroenterology 2011) grown in media called WENR+inhibitors. RNA was also isolated from various steps in the culturing and differentiation protocol.

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Deep Visual Proteomics advances human colon organoid models by revealing a switch to an in vivo-like phenotype upon xenotransplantation

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