Abstract
The development of alternative in vitro culture methods has increased in the last decade as three-dimensional organoids of various tissues, including those of the small and large intestines. Due to their multicellular composition, organoids offer advantages over traditionally used immortalized or primary cell lines. However, organoids must be accurate models of their tissues of origin. This study compared gene expression profiles with respect to markers of specific cell types (stem cells, enterocytes, goblet, and enteroendocrine cells) and barrier maturation (tight junctions) of colonoid and enteroid cultures with their tissues of origin and colonoids with enteroids. Colonoids derived from three healthy pigs formed multilobed structures with a monolayer of cells similar to the crypt structures in colonic tissue. Colonoid and enteroid gene expression signatures were more similar to those found for the tissues of their origin than to each other. However, relative to their derived tissues, organoids had increased gene expression levels of stem cell markers Sox9 and Lgr5 encoding sex-determining region Y-box 9 and leucine-rich repeat-containing G protein-coupled rector 5, respectively. In contrast, expression levels of Occl and Zo1 encoding occludin and zonula occludens 1, respectively, were decreased. Expression levels of the cell lineage markers Atoh1, Cga, and Muc2 encoding atonal homolog 1, chromogranin A, and mucin 2, respectively, were decreased in colonoids, whereas Sglt1 and Apn encoding sodium-glucose transporter 1 and aminopeptidase A, respectively, were decreased in enteroids. These results indicate colonoid and enteroid cultures were predominantly comprised of undifferentiated cell types with decreased barrier maturation relative to their tissues of origin.
Highlights
There are differences in the structure and function of the two distinct anatomical sections of the intestinal tract, the small and large intestine [1]
Before isolation of the crypts from ileum tissue, the villus compartments were removed from the epithelial surface through scraping, to reduce the harvesting of undesirable differentiated cell types that would hamper the development of enteroids
MRNA expression levels of Occl and zonula occludens 1 (Zo1) were lower, whereas Cldn was significantly elevated (4.2-fold ± 0.9-fold) in colonoids relative to the colon (Fig. 6). These results indicate that the stem cell niche of colonoid cultures is being maintained but that secretory cell lineages are decreased relative to the tissue from which they were derived, resulting in predominantly immature, undifferentiated structures
Summary
There are differences in the structure and function of the two distinct anatomical sections of the intestinal tract, the small and large intestine [1]. The large intestine has crypt domains that extend into the submucosa, villi are absent [1]. The small intestinal villi increase the surface area, functionally increasing the absorption of readily available nutrients. Other differentiated cell types are predominantly found in the villi of the small intestine and the upper crypt region and surface epithelium of the colon [3, 4]. Enteroendocrine cells are present in both segments but represent less than 1% of epithelial cells [1] Together, these epithelial cells form the intrinsic barrier, of which junctional proteins such as occludin, claudin, and zonula occludens are important components [10] that regulate barrier function, controlling the entry of luminal nutrients, ions, and water [11]
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