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Detailed survey of an in vitro intestinal epithelium model by single-cell transcriptomics.

ABSTRACT: The co-culture of two adult human colorectal cancer cell lines, Caco-2 and HT29, on Transwell is commonly used as an in vitro gut mimic, yet the translatability of insights from such a system to adult human physiological contexts is not fully characterized. Here, we used single-cell RNA sequencing on the co-culture to obtain a detailed survey of cell type heterogeneity in the system and conducted a holistic comparison with human physiology. We identified the intestinal stem cell-, transit amplifying-, enterocyte-, goblet cell-, and enteroendocrine-like cells in the system. In general, the co-culture was fetal intestine-like, with less variety of gene expression compared to the adult human gut. Transporters for major types of nutrients were found in the majority of the enterocytes-like cells in the system. TLR 4 was not expressed in the sample, indicating that the co-culture model is incapable of mimicking the innate immune aspect of the human epithelium.

SUBMITTER: Ran R

PROVIDER: S-EPMC10959667 | biostudies-literature | 2024 Apr

REPOSITORIES: biostudies-literature

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Detailed survey of an <i>in vitro</i> intestinal epithelium model by single-cell transcriptomics.

Ran Ran R Muñoz Briones Javier J Jena Smrutiti S Anderson Nicole L NL Olson Matthew R MR Green Leopold N LN Brubaker Douglas K DK

iScience 20240301 4

The co-culture of two adult human colorectal cancer cell lines, Caco-2 and HT29, on Transwell is commonly used as an <i>in vitro</i> gut mimic, yet the translatability of insights from such a system to adult human physiological contexts is not fully characterized. Here, we used single-cell RNA sequencing on the co-culture to obtain a detailed survey of cell type heterogeneity in the system and conducted a holistic comparison with human physiology. We identified the intestinal stem cell-, transit ...[more]

PMID: 38523788

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Project description:The gut plays a critical role in maintaining human health by facilitating the absorption of nutrients, regulating metabolism, and interacting with the immune system and gut microbiota. The co-culture of two human colorectal cancer cell lines, Caco-2 and HT29, on Transwell is commonly used as an in vitro gut mimic in studies of intestine absorption pharmacokinetic, gut mechanics, and gut-microbes interplay given the similar morphology, expression of transporters and enzymes, and barrier function. However, to sufficiently evaluate the translatability of insights from such a system to human physiological contexts, a detailed survey of cell type heterogeneity in the system and a holistic comparison with human physiology needs to be conducted rather than by the presence of a few well-studied proteins. Single-cell RNA sequencing provides high-resolution expression profiles of cells in the co-culture, enabling the heterogeneity to be characterized and the similarity to human epithelial cells to be evaluated. 16019 genes transcriptional profile in 13784 cells were acquired and compared to human epithelial cells (GSE185224). We identified the intestinal stem cell, transit amplifying, enterocyte, goblet cell, and enteroendocrine-like cells together with differentiating HT29 cells in the system based on the expression of canonical markers in healthy adult human epithelial cells. The epithelium-like co-culture was fetal intestine-like, with less variety of gene expression compared to the human gut. Transporters for major types of substance (lipid, amino acid, ion, water, etc.) were found transcribed in the majority of the enterocytes-like cells in the system. However, some of the well-studied transporters were absent. Toll-like receptors were not highly expressed in the sample, yet the treatment of LPS still caused a mild change in TEER and gene expression, possibly by the interaction with CD14. Overall, the Caco-2/HT29 co-culture is a cost-effective epithelium model for drug permeability testing or mechanical simulation, but its discrepancy with the real epithelium phenotype-wise is not negligible. As a result, its response to biological factors might not provide transferrable knowledge to the study of human gut physiology, especially the innate immune aspect.

Project description:Background & aimsSingle-cell transcriptomics offer unprecedented resolution of tissue function at the cellular level, yet studies analyzing healthy adult human small intestine and colon are sparse. Here, we present single-cell transcriptomics covering the duodenum, jejunum, ileum, and ascending, transverse, and descending colon from 3 human beings.MethodsA total of 12,590 single epithelial cells from 3 independently processed organ donors were evaluated for organ-specific lineage biomarkers, differentially regulated genes, receptors, and drug targets. Analyses focused on intrinsic cell properties and their capacity for response to extrinsic signals along the gut axis across different human beings.ResultsCells were assigned to 25 epithelial lineage clusters. Multiple accepted intestinal stem cell markers do not specifically mark all human intestinal stem cells. Lysozyme expression is not unique to human Paneth cells, and Paneth cells lack expression of expected niche factors. Bestrophin 4 (BEST4)+ cells express Neuropeptide Y (NPY) and show maturational differences between the small intestine and colon. Tuft cells possess a broad ability to interact with the innate and adaptive immune systems through previously unreported receptors. Some classes of mucins, hormones, cell junctions, and nutrient absorption genes show unappreciated regional expression differences across lineages. The differential expression of receptors and drug targets across lineages show biological variation and the potential for variegated responses.ConclusionsOur study identifies novel lineage marker genes, covers regional differences, shows important differences between mouse and human gut epithelium, and reveals insight into how the epithelium responds to the environment and drugs. This comprehensive cell atlas of the healthy adult human intestinal epithelium resolves likely functional differences across anatomic regions along the gastrointestinal tract and advances our understanding of human intestinal physiology.

Dataset Information

Detailed survey of an in vitro intestinal epithelium model by single-cell transcriptomics.

Publications

Detailed survey of an <i>in vitro</i> intestinal epithelium model by single-cell transcriptomics.

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