Chicken-derived RSPO1 and WNT3 contribute to maintaining longevity of chicken intestinal organoid cultures

Intestinal organoids are advanced cellular models, which are widely used in mammalian studies to mimic and study in vivo intestinal function and host-pathogen interactions. Growth factors WNT3 and RSPO1 are crucial for the growth of intestinal organoids. Chicken intestinal organoids are currently cultured with mammalian WNT3a and RSPO1, however, maintaining their longevity has shown to be challenging. Based on the limited homology between mammalian and avian RSPO1, we expect that chicken-derived factors are required for the organoid cultures. We examined the effect of chicken-specific WNT3 and RSPO1 in combination with Prostaglandin E2 and a Forkhead box O1-inhibitor on the development of chicken intestinal organoids. Isolated crypts from embryonic tissue of laying hens were successfully cultured for 15 passages in the presence of chicken-derived WNT3 and RSPO1, whereas no growth was observed when mammalian factors were used. Furthermore, stem cells maintained their ability to differentiate into goblets, enterocytes, and enteroendocrine cells for the 3D and 2D structures. The chicken intestinal organoids can be an important tool to study responses of the intestinal epithelium to pathogens, drugs or feed additives.