(B-73) Detailed Survey of an in-vitro Intestinal Epithelium Model by Single-Cell Transcriptomics

Introduction:: The intestine 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. Co-culture of two human colorectal cancer cell lines, Caco-2 and HT29, on a Transwell insert forms a monolayer that is found to have similar morphology, expression of several transporters and enzymes, and barrier function with the human gut epithelium, making it commonly used as an in-vitro gut mimic in studies of intestine absorption pharmacokinetic, gut mechanics, and gut-microbes interplay. However, a detailed survey of cell heterogeneity in the system and a holistic comparison with human physiology needs to be conducted to sufficiently evaluate the translatability of insights from such an experimental model into human physiological contexts. Single-cell RNA sequencing (scRNA-seq) 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.



Materials and Methods:: Caco-2 (ATCC, passage no. 8) and HT29 (ATCC, passage No.13) were cultured separately at 1x10⁵ cells/ cm² and seeded together into the apical chamber of three Transwell (Corning Inc, Costar, USA) with a ratio of 9:1 on day 0. Two Transwells were used as controls, where cells were co-cultured and harvested on day 23. The other transwell was treated with 1 ng/ml Escherichia coli O111:B4 lipopolysaccharides (LPS) on day 22 and harvested on day 23. Transepithelial electrical resistance (TEER) was measured in both conditions by an epithelial volt-ohmmeter (EVOM2; WPI, Berlin, Germany) with STX2 Chopstick probes to determine the cell monolayer integrity. Cells were prepared for scRNA-seq following the manufacturer’s instructions (10X Genomics). 16,019 genes in 13784 cells were acquired after quality control and normalization. Cell subsets were identified, characterized, and compared to the features of healthy adult human epithelial cells (GSE185224).


Results, Conclusions, and Discussions:: The Caco-2/HT29 co-culture system exhibits high heterogeneity and resembles the fetal small intestine, with a less diverse gene expression profile compared to the healthy adult human gut. We identified intestinal stem cell-like cells, transit amplifying cell-like cells, enterocyte-like cells, goblet cell-like cells, and enteroendocrine-like cells, along with differentiating HT29 cells, in the system based on the expression of canonical markers in healthy adult human epithelial cells or monoculture HT29 (Figure 1). Although most Wnt ligands were not actively transcribed (Figure 2) in the model, the activities of Wnt downstream genes like ASCL2 (Figure 1C) and Wnt inhibitors like PLA2G2A (Figure 1D) suggest pathway activation, which could be attributed to the intrinsic APC mutation in these two colorectal cancer cell lines. Nutrient transporters were transcribed in most enterocyte-like cells in the system, but the absence of some well-studied transporters suggests a reprogrammed metabolism (Figure 3). Toll-like receptor 4 (TLR4), the receptor responsible for recognizing and responding to lipopolysaccharide (LPS) on the outer membrane of Gram-negative bacteria, was not found to be expressed in the sample. Nevertheless, LPS treatment still induced a mild change in TEER and gene expression, possibly due to the potential interaction of LPS with TLR4's co-receptor CD14, which was transcribed in the system at a very low level (Figure 4).

In conclusion, the Caco-2/HT29 co-culture system is a cost-effective epithelium model for drug permeability testing or mechanical simulation, but its discrepancies with the real epithelium cannot be neglected.



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