Initial pig developmental stage influences intestinal organoid growth but not cellular composition

Abstract

Background: Intestinal organoids are promising tools in the context of animal experiment reduction but a thorough characterization of the impact of the origin of intestinal stem cells (ISC) on organoid phenotype is needed to routinely use this cellular model. Our objective was to evaluate the effect of ISC donor age on the growth, morphology and cellular composition of intestinal organoids derived from pig.

Methods: Organoids were derived from jejunal and colonic ISC obtained from 1-, 7-, 28-, 36-and 180-day-old pigs and passaged three times.

Results: We first confirmed by qPCR that the expression of 18% of the >80 studied genes related to various intestinal functions differed between jejunal and colonic organoids after two passages (p < 0.05). Growth and morphology of organoids depended on intestinal location (greater number and larger organoids derived from colonic than jejunal ISC, p < 0.05) but also pig age. Indeed, when ISC were derived from young piglets, the ratio of organoids to spheroids was greater (p < 0.05), spheroids were larger during the primary culture but smaller after two passages (p < 0.05) and organoids were smaller after one passage (p > 0.05) compared to ISC from older pigs. Finally, no difference in cellular composition, evaluated by immunostaining of markers of the major intestinal cell types (absorptive, enteroendocrine and goblet cells) was observed between organoids originating from 7-or 180-day-old pigs, but differences between intestinal site origins were noticed.

Conclusion: In conclusion, while the age of the tissue donor affected organoid growth and morphology, it did not influence the phenotype.

Keywords

colonoid / development / enteroid / organoid phenotype / piglet

References

[1] Roura E, Koopmans S-J, Lallès J-P, et al. Critical review evaluating the pig as a model for human nutritional physiology. Nutr Res Rev. 2016;29:60-90.

[2] Mussard E, Lencina C, Boudry G, et al. Culture of piglet intestinal 3D organoids from cryopreserved epithelial crypts and establishment of cell monolayers. JoVE. 2023;192:64917.

[3] Barnett AM, Mullaney JA, Hendriks C, Le Borgne L, McNabb WC, Roy NC. Porcine colonoids and enteroids keep the memory of their origin during regeneration. Am J Physiol-Cell Physiol. 2021;320:C794-C805.

[4] Beaumont M, Lencina C, Painteaux L, et al. A mix of functional amino acids and grape polyphenols promotes the growth of piglets, modulates the gut microbiota in vivo and regulates epithelial homeostasis in intestinal organoids. Amino Acids. 2022;54:1357-1369.

[5] Wang J, Zhao Y, Cui T, et al. AhR ligands from LGG metabolites promote piglet intestinal ILC3 activation and IL-22 secretion to inhibit PEDV infection. J Virol. 2024;98:e0103924.

[6] Vermeire B, Gonzalez LM, Jansens RJJ, Cox E, Devriendt B. Porcine small intestinal organoids as a model to explore ETEC-host interactions in the gut. Vet Res. 2021;52:94.

[7] Gelberg HB. Comparative anatomy, physiology, and mechanisms of disease production of the esophagus, stomach, and small intestine. Toxicol Pathol. 2014;42:54-66.

[8] Schaaf CR, Polkoff KM, Carter A, et al. A LGR5 reporter pig model closely resembles human intestine for improved study of stem cells in disease. FASEB J. 2023;37:e22975.

[9] Gonzalez LM, Williamson I, Piedrahita JA, Blikslager AT, Magness ST. Cell lineage identification and stem cell culture in a porcine model for the study of intestinal epithelial regeneration. Singh SR, editor. PLoS One. 2013;8:e66465.

[10] Clevers HC, Bevins CL. Paneth cells: maestros of the small intestinal crypts. Annu Rev Physiol. 2013;75:289-311.

[11] Moeser AJ, Klok CV, Ryan KA, et al. Stress signaling pathways activated by weaning mediate intestinal dysfunction in the pig. American journal of physiology-gastrointestinal and liver. Phys Ther. 2007;292:G173-G181.

[12] Boudry G, Péron V, Le Huërou-Luron I, Lallès JP, Sève B. Weaning induces both transient and long-lasting modifications of absorptive, secretory, and barrier properties of piglet intestine. J Nutr. 2004;134:2256-2262.

[13] Liu Y, Zheng Z, Yu L, et al. Examination of the temporal and spatial dynamics of the gut microbiome in newborn piglets reveals distinct microbial communities in six intestinal segments. Sci Rep. 2019;9:3453.

[14] Mussard E, Lencina C, Gallo L, et al. The phenotype of the gut region is more stably retained than developmental stage in piglet intestinal organoids. Front Cell Dev Biol. 2022;10:983031.

[15] Arnaud AP, Rome V, Richard M, Formal M, David-Le Gall S, Boudry G. Post-natal co-development of the microbiota and gut barrier function follows different paths in the small and large intestine in piglets. FASEB J. 2020;34:1430-1446.

[16] Verdile N, Mirmahmoudi R, Brevini TAL, Gandolfi F. Evolution of pig intestinal stem cells from birth to weaning. Animal. 2019;13:2830-2839.

[17] Beaumont M, Lencina C, Fève K, et al. Disruption of the primocolonizing microbiota alters epithelial homeostasis and imprints stem cells in the colon of neonatal piglets. FASEB J. 2023;37:e23149.

[18] Charton E, Bourgeois A, Bellanger A, et al. Infant nutrition affects the microbiota-gut-brain axis: comparison of human milk vs. infant formula feeding in the piglet model. Front Nutr. 2022;9:976042.

[19] Beaumont M, Mussard E, Barilly C, et al. Developmental stage, solid food introduction, and suckling cessation differentially influence the Comaturation of the gut microbiota and intestinal epithelium in rabbits. J Nutr. 2022;152:723-736.

[20] Paës C, Gidenne T, Bébin K, et al. Early Introduction of Plant Polysaccharides Drives the Establishment of Rabbit Gut Bacterial Ecosystems and the Acquisition of Microbial Functions. mSystems. 2022;7:e00243-22.

[21] Ulshen MH, Lecce JG, Stiles AD, Lyn-Cook LE. Effects of nursing on growth and development of small bowel mucosa in newborn piglets. Pediatr Res. 1991;30:337-341.

[22] Sheard NF, Walker WA. The role of breast Milk in the development of the gastrointestinal tract. Nutr Rev. 2009;46:1-8.

[23] Shirkey TW, Siggers RH, Goldade BG, et al. Effects of commensal bacteria on intestinal morphology and expression of Proinflammatory cytokines in the Gnotobiotic pig. Exp Biol Med (Maywood). 2006;231:1333-1345.

[24] Nalapareddy K, Zheng Y, Geiger H. Aging of intestinal stem cells. Stem Cell Reports. 2022;17:734-740.

[25] Itzkovitz S, Blat IC, Jacks T, Clevers H, van Oudenaarden A. Optimality in the development of intestinal crypts. Cell. 2012;148:608-619.

[26] Nelson MR, Howard D, Jensen OE, King JR, Rose FRAJ, Waters SL. Growth-induced buckling of an epithelial layer. Biomech Model Mechanobiol. 2011;10:883-900.

[27] Broguiere N, Isenmann L, Hirt C, et al. Growth of epithelial organoids in a defined hydrogel. Adv Mater. 2018;30:1801621.

[28] Thalheim T, Siebert S, Quaas M, et al. Epigenetic drifts during long-term intestinal organoid culture. Cells. 2021;10:1718.

[29] Noblet J, Etienne M. Estimation of sow Milk nutrient output. J Anim Sci. 1989;67:3352-3359.

[30] Petersen V. The development of feeding and investigatory behaviour in free-ranging domestic pigs during their first 18 weeks of life. Appl Anim Behav Sci. 1994;42:87-98.

[31] Beaumont M, Cauquil L, Bertide A, et al. Gut microbiota-derived metabolite signature in suckling and weaned piglets. J Proteome Res. 2021;20:982-994.

[32] Dougherty MW, Kudin O, Mühlbauer M, Neu J, Gharaibeh RZ, Jobin C. Gut microbiota maturation during early human life induces enterocyte proliferation via microbial metabolites. BMC Microbiol. 2020;20:205.

[33] Kaiko GE, Ryu SH, Koues OI, et al. The colonic crypt protects stem cells from microbiota-derived metabolites. Cell. 2016;165:1708-1720.

[34] Pearce SC, Weber GJ, Van Sambeek DM, Soares JW, Racicot K, Breault DT. Intestinal enteroids recapitulate the effects of short-chain fatty acids on the intestinal epithelium. PLoS One. 2020;15:e0230231.

[35] Sahoo DK, Borcherding DC, Chandra L, et al. Differential transcriptomic profiles following stimulation with lipopolysaccharide in intestinal organoids from dogs with inflammatory bowel disease and intestinal mast cell tumor. Cancer. 2022;14:3525.

RIGHTS & PERMISSIONS

2024 The Author(s). Animal Models and Experimental Medicine published by John Wiley & Sons Australia, Ltd on behalf of The Chinese Association for Laboratory Animal Sciences.