Regional epithelial cell diversity in the small intestine of pigs

Epithelial cell RNA expression was analyzed across multiple locations of the small intestine in pigs, revealing regional specialization of cells, including differences in localization and transcriptional profiles. Understanding regional distribution and specialization of small intestinal epithelial cells is crucial for developing methods to control appetite, stress, and nutrient uptake in swine. To establish a better understanding of specific epithelial cells found across different regions of the small intestine in pigs, we utilized single-cell RNA sequencing (scRNA-seq) to recover and analyze epithelial cells from duodenum, jejunum, and ileum. Cells identified included crypt cells, enterocytes, BEST4 enterocytes, goblet cells, and enteroendocrine (EE) cells. EE cells were divided into two subsets based on the level of expression of the EE lineage commitment gene, NEUROD1. NEUROD1(hi) EE cells had minimal expression of hormone-encoding genes and were dissimilar to EE cells in humans and mice, indicating a subset of EE cells unique to pigs. Recently discovered BEST4 enterocytes were detected in both crypts and villi throughout the small intestine via in situ staining, unlike in humans, where BEST4 enterocytes are found only in small intestinal villi. Proximal-to-distal gradients of expression were noted for hormone-encoding genes in EE cells and nutrient transport genes in enterocytes via scRNA-seq, demonstrating regional specialization. Regional gene expression in EE cells and enterocytes was validated via quantitative PCR (qPCR) analysis of RNA isolated from epithelial cells of different small intestinal locations. Though many genes had similar patterns of regional expression when assessed by qPCR of total epithelial cells, some regional expression was only detected via scRNA-seq, highlighting advantages of scRNA-seq to deconvolute cell type-specific regional gene expression when compared to analysis of bulk samples. Overall, results provide new information on regional localization and transcriptional profiles of epithelial cells in the pig small intestine. Lay Summary Cells lining the intestinal tract (i.e., epithelial cells) provide a barrier to the outside environment but also play important specialized roles in nutrient absorption and secretion of mucus or hormones involved in controlling appetite and digestion. While similar cell types can be found throughout the small intestine, they have even more specialized function depending on region of the small intestine. Identification and characterization of intestinal epithelial cells are foundational to promoting pig intestinal health for optimal growth. Our research identified six types of epithelial cells across the small intestine of pigs. Enterocytes, an absorptive cell type, shared commonalities with human enterocytes, but a population of enteroendocrine cells, which secrete hormones, was unique to pigs. The location of certain epithelial cells in the intestine was identified and informed the relationship between various epithelial cell types. Overall, a clearer understanding of specific epithelial cells in the porcine intestine is provided, proving a critical foundation to further research aimed at maximizing pig intestinal health.