Abstract
The intestine is composed of an epithelial layer containing rapidly proliferating cells that mature into two regions, the small and the large intestine. Although previous studies have identified stem cells as the cell-of-origin for intestinal epithelial cells, no studies have directly compared stem cells derived from these anatomically distinct regions. Here, we examine intrinsic differences between primary epithelial cells isolated from human fetal small and large intestine, after in vitro expansion, using the Wnt agonist R-spondin 2. We utilized flow cytometry, fluorescence-activated cell sorting, gene expression analysis and a three-dimensional in vitro differentiation assay to characterize their stem cell properties. We identified stem cell markers that separate subpopulations of colony-forming cells in the small and large intestine and revealed important differences in differentiation, proliferation and disease pathways using gene expression analysis. Single cells from small and large intestine cultures formed organoids that reflect the distinct cellular hierarchy found in vivo and respond differently to identical exogenous cues. Our characterization identified numerous differences between small and large intestine epithelial stem cells suggesting possible connections to intestinal disease.
Highlights
The intestine consists of two major subdivisions: the small intestine (SI) and the large intestine (LI), which differ in structure and function
We found that growth of normal fetal intestinal cells on the feeder layer required the addition of R-spondin 2, a Wnt agonist acting via the leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5) receptor [8,19]
After primary cell expansion in vitro, flow cytometric analyses revealed enrichment of epithelial cells, as marked by Epithelial Cell Adhesion Molecule (EPCAM), from 21.6 to 92.3% in cells expanded from SI and from 34.3% to 92.4% in cells expanded from LI (Fig. 1C&D)
Summary
The intestine consists of two major subdivisions: the small intestine (SI) and the large intestine (LI), which differ in structure and function. The SI is largely responsible for the digestion and absorption of food while the LI aids in final water absorption and waste removal. Wnt and Notch control the well-defined epithelial hierarchy in the intestine, helping to maintain stem cell homeostasis. Since these pathways require receptors, ligands and transcriptional regulation, it is unclear whether differences observed between the SI and LI are primarily due to intrinsic or extrinsic mechanisms [1,2]. A thorough investigation of the origin of the differences between the SI and LI has yet to be done
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