Detailed Survey of an in-vitro Intestinal Epithelium Model by Single-Cell Transcriptomics
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ABSTRACT:
PROVIDER: PRJNA976932 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR24753652_1.fastq.gz | Fastqsanger.gz | |||
| SRR24753652_2.fastq.gz | Fastqsanger.gz | |||
| SRR24753653_1.fastq.gz | Fastqsanger.gz | |||
| SRR24753653_2.fastq.gz | Fastqsanger.gz | |||
| SRR24753654_1.fastq.gz | Fastqsanger.gz |
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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.
2023-05-31 | GSE233628 | GEO
Project description: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.
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Project description:The small intestine is responsible for nutrient absorption and it is one of the most important interfaces between the environment and our body. During aging, changes in the structure of the epithelium lead to food malabsorption and reduced barrier function thus increasing disease risk in aging. The molecular drivers of these alterations remain poorly understood. Here, we compared the proteomes of small intestinal crypts from mice across different anatomical regions and age groups. We found that aging alters epithelial immune responses, metabolic networks and stem cell proliferation, and it is accompanied by a region-dependent skewing in the cellular composition of the intestinal epithelium. Of note, a short period of dietary restriction followed by re-feeding partially restores the epithelium to a youthful state by promoting the differentiation of intestinal stem cells (ISCs) towards the secretory lineage. Using in vitro and in vivo studies, we identify Hmgcs2 (3-hydroxy-3-methylglutaryl-CoA synthetase 2) – the rate limiting enzyme in the synthesis of ketone bodies – as a modulator of ISCs differentiation, which responds to dietary changes. This study provides an atlas of age-dependent proteome changes in defined regions of the intestinal epithelium and characterizes how young and old mice adapt to drastic changes of diet, such as dietary restriction and re-feeding.
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Project description:Background and Aims: Single-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 humans. Methods: 12,590 single epithelial cells from three 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 capacity for response to extrinsic signals along the gut axis across different humans. Results: Cells were assigned to 25 epithelial lineage clusters. Human intestinal stem cells (ISCs) are not specifically marked by many murine ISC markers. Lysozyme expression is not unique to human Paneth cells (PCs), and PCs lack expression of expected niche-factors. BEST4+ cells express NPY and show maturational differences between SI 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-junction, and nutrient absorption genes show unappreciated regional expression differences across lineages. Differential expression of receptors and drug targets across lineages reveals biological variation and potential for variegated responses. Conclusions: Our 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 anatomical regions along the gastrointestinal tract and advances our understanding of human intestinal physiology.
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Project description:A proximal-to-distal survey of healthy adult human small intestine and colon epithelium by single-cell transcriptomics
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Project description:Intestinal epithelial cells absorb nutrients, respond to microbes, function as a barrier and help to coordinate immune responses. Here we report profiling of 53,193 individual epithelial cells from the small intestine and organoids of mice, which enabled the identification and characterization of previously unknown subtypes of intestinal epithelial cell and their gene signatures. We found unexpected diversity in hormone-secreting enteroendocrine cells and constructed the taxonomy of newly identified subtypes, and distinguished between two subtypes of tuft cell, one of which expresses the epithelial cytokine Tslp and the pan-immune marker CD45, which was not previously associated with non-haematopoietic cells. We also characterized the ways in which cell-intrinsic states and the proportions of different cell types respond to bacterial and helminth infections: Salmonella infection caused an increase in the abundance of Paneth cells and enterocytes, and broad activation of an antimicrobial program; Heligmosomoides polygyrus caused an increase in the abundance of goblet and tuft cells. Our survey highlights previously unidentified markers and programs, associates sensory molecules with cell types, and uncovers principles of gut homeostasis and response to pathogens.
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Project description:We obtained a spatial measurement of RNA and Proteins in the small intestinal epithelium along the crypt-villus axis. We found that both were spatially heterogeneous, yet often spatially anti-correlated. We developed a Bayesian approach to infer protein translation and degradation rates from the combined spatial profiles, and demonstrate that space-independent protein-synthesis delays can explain the mRNA-protein discordances. Our work provides a proteomic spatial blueprint of the Intestinal epithelium and highlights the importance of protein measurements for inferring states of tissue cells that operate outside of steady state
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