Modeling age-dependent developmental changes in the expression of genes involved in citrulline synthesis using pig enteroids

Authors: MOHAMMAD, MAHMOUD - Children'S Nutrition Research Center (CNRC); DIDELIJA, INKA - Children'S Nutrition Research Center (CNRC); STOLL, BARBARA - Children'S Nutrition Research Center (CNRC); Burrin, Douglas - Doug; MARINI, JUAN - Children'S Nutrition Research Center (CNRC)

Submitted to: Physiological Reports
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/7/2020
Publication Date: 11/12/2020
Citation: Mohammad, M., Didelija, I., Stoll, B., Burrin, D.G., Marini, J. 2020. Modeling age-dependent developmental changes in the expression of genes involved in citrulline synthesis using pig enteroids. Physiological Reports. 8(21):e14565. https://doi.org/10.14814/phy2.14565.
DOI: https://doi.org/10.14814/phy2.14565

Interpretive Summary: The isolation and culture of stem cells from the small intestine results in a novel model called enteroids. These are 3-D structures that resemble the functional unit of the small intestine. It has been shown that stem cells have a "location" memory and that when they develop into enteroids they "remember" from which section of the small intestine they originated. Here we tested if intestinal stem cells have "developmental" memory, this is they "remember" at what age they were collected. We harvested small intestine from pigs from different ages, from preterm piglets to post-weaning animals, isolated stem cells, and developed enteroids. We looked at different developmental markers but we were not able to find evidence of "developmental" memory. It is likely that extracellular cues may be needed to reproduce the age-dependent phenotype.

Technical Abstract: Age-dependent changes in the intestinal gene expression of enzymes involved in the metabolism of citrulline and arginine are well characterized. Enteroids, a novel ex-vivo model that recreates the 3D structure of the intestinal crypt-villus unit, have shown to replicate molecular and physiological profiles of the intestinal segment from where they originated ('location memory'). The present study tested the hypothesis that enteroids recapitulate the developmental changes observed in vivo regarding citrulline production in pigs ('developmental memory'). Preterm (10- and 5-d preterm) and term pigs at birth, together with 7- and 35-d-old pigs were studied. Gene expression was measured in jejunal samples and in enteroids derived from this segment. Whole body citrulline production was measured by isotope dilution and enteroid citrulline production by accumulation in the media. With the exception of arginase I and inducible nitric oxide synthase, all the genes investigated expressed in jejunum were expressed by enteroids. In the jejunum, established markers of development (lactase and sucrase-isomaltase), as well as genes that code for enzymes involved in the production and utilization of citrulline and arginine, underwent the ontogenic changes described in the literature. However, enteroid expression of these genes, as well as citrulline production, failed to recapitulate the changes observed in vivo. Under culture conditions used in our study enteroids derived from jejunal crypts of pigs at different ages failed to replicate the gene expression observed in whole tissue and whole body citrulline production. Additional extracellular cues may be needed to reproduce the age-dependent phenotype.