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

Abstract

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. Isolated crypts from embryonic tissue of laying hens were growing in the presence of chicken WNT3 and RSPO1, whereas growth in the presence of mammalian Wnt3a and Rspo1 was limited. Moreover, the growth was increased by using Prostaglandin E2 (PGE2) and a Forkhead box O1-inhibitor (FOXO1-inhibitor), allowing to culture these organoids for 15 passages. Furthermore, stem cells maintained their ability to differentiate into goblets, enterocytes and enteroendocrine cells in 2D structures. Overall, we show that chicken intestinal organoids can be cultured for multiple passages using chicken-derived WNT3 and RSPO1, PGE2, and FOXO1-inhibitor.

Differential Salmonella Typhimurium intracellular replication and host cell responses in caecal and ileal organoids derived from chicken

“…They were cultivated in 3D in Matrigel™ matrix and exposed to a probiotic, a TLR2 ligand [ 9 ] or various chemicals [ 10 ] to demonstrate a functional epithelium. Many studies have described optimization in the culture conditions with different concentrations of EGF, R-Spondin and of Noggin to improve enrichments, passages and cryopreservation of chicken organoids [ 11 – 13 ]. Two novel methods of cultivation of intestinal chicken organoids were described in order to allow access to the apical side of the cells: the two-dimensional (2D) polarized models of intestinal monolayers and the inside-out organoids [ 14 – 16 ].…”

Temporal transcriptome profiling of floating apical out chicken enteroids suggest stability and reproducibility

“…Despite the capability existing to consecutively passage gel-embedded chicken enterospheres (organoid nomenclature for the poorly-differentiated spherical structures observed using this technique), currently there is no evidence for the ability to passage floating enteroid cultures [ 13 , 14 , 16 , 46 , 47 ]. Various trials have been performed in the floating chicken enteroids, but continued bud growth after passage has not been recognised [ 16 ].…”

Lactiplantibacillus plantarum and Saccharomyces cerevisiae—Fermented Coconut Water Alleviates Dextran Sodium Sulfate-Induced Enteritis in Wenchang Chicken: A Gut Microbiota and Metabolomic Approach

“…Following the protocol described by the method of O. Srinual for staining chick intestinal tissue [17], the cecal tissue was fixed in 4% paraformaldehyde (w/v) for 24 h. Subsequently, it was dehydrated, embedded in paraffin, and cut into 3 µm sections. Some colon samples were stained with hematoxylin and eosin (H&E) as described earlier [18][19][20][21]. Tissue pathology was assessed using optical microscopy (Nikon Corporation, Japan), and histological injury scores were calculated to evaluate the extent of tissue damage and analyzed using Image-Pro Plus 6.0 analysis software, with results reported in millimeters.…”