Chicken-derived R-spondin1, Wnt3a, Nrg1 and human FGF1 allow branched growth of chicken intestinal organoid cultures.

Intestinal organoids are widely used in mammalian studies to mimic and study intestinal function and host-pathogen interactions without using of whole animal studies. For chicken, this was not yet feasible. Recently, we have shown that embryonal intestinal organoids can grow for 10-15 passages (approximately 6 weeks) when using chicken-derived Rspo1 and Wnt3. In this paper, we show that replacing Wnt3 with chicken Wnt3a and adding chicken Nrg1 and human FGF1 results in efficient and long lasting growth, over 40 weeks, of embryonal chicken intestinal organoids. Moreover, the typical branching phenotype, which is normally observed for mammalian species such as mouse and pig intestinal organoids, is now also observed in the chicken organoids when Rspo1, Wnt3a, Nrg1 and hFGF1 are used. This branching is enhanced by inhibiting FoxO1 with AS1842856, resulting in an increase of growth. Moreover, we show the presence of Lgr5 positive stem cells, goblets, endocrine cells, Paneth cells and enterocytes. In agreement with the observed presence of goblet cells, we observed mucus secretion in the 2D cultures. This new and improved culture condition for chicken intestinal organoids allows for in vitro research that can partially replace animal testing with respect to, for instance, replacement of antibiotics, feed additives and drug development.