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.

Figure 1

Schematic overview of the isolation and culture conditions of chicken intestinal organoid. (1) Isolation of small and large intestines (2) Intestinal tissue was cut into tiny segments and washed with PBS-EDTA. (3) To obtain a homogenous suspension, segments were minced using a Potter–Elvehjem PTFE pestle and glass tube. (4) After multiple centrifugation steps, the pellet was resuspended in ice-cold Matrigel (5) and seeded in a culture plate (6). After polymerization of Matrigel, crypts were cultured with indicated media as defined in supplementary Table 1. (7) Monolayer cultures were obtained from three-dimensional cultures.

Figure 2

Sequence alignments of (a) WNT3 protein for mouse, human and chickens. Chicken vs mouse and chicken vs human are 96% identical (human vs mouse: 98%). (b) Alignment of RSPO1 for mouse, human and chicken. Chicken and mouse is 65% identical and chicken and human is 68% (human and mouse: 89%). Non-consensus amino acids (grey).

Figure 3

Growth factors RSPO1 and WNT3, from murine or chicken origin, are important for establishing intestinal organoid growth. (a) Immunoblot analysis of chicken RSPO1 and chicken WNT3 protein expression in transfected Hek293t cells. Actin was used as the protein loading control and anti-flag HRP was used to visualize RSPO1 and WNT3. Empty Vector (E.V.) is taken along as a control. (b) Brightfield images of mouse intestinal organoids (I,II) and chicken intestinal organoids (III,IV) at passage 2 (5 days after passage). Cultured in Matrigel with ICM, supplemented with Rspo1 and Wnt3 of murine origin, (c) or supplemented with chicken-derived RSPO1 and WNT3. Images and immunoblot are representative of three independent cultures. Scale bar 200 µm.

Figure 4

Supplementary compounds for organoid growth. Brightfield images of embryonic chicken organoid cultured in Matrigel with BCM of passage 1, 1,2 and 3 days after splitting. (a,e,i) with additional RSPO1 and WNT3 of chicken origin. (b,f,j) with additional RSPO1/WNT3 of chicken origin and 0.2 µM Forkhead box O1-inhibitor (FOXO1-i). (c,g,k) with additional RSPO1/WNT3 and 2.5 µg/ml prostaglandin E2 (PGE2) (d,h,l). With all 4 supplemented growth factors (WNT3/RSPO1/PGE2/FoxO1-i). Images are representative of three independent cultures. Scale bar: 1000 µm. (m) Diameter of organoids when culturing with different supplements, were measured manually and plotted in Graphpad Prism. Four outliers were excluded after performing an outlier test in Graphpad Prism. Statistics were performed using a Kruskal–Wallis test followed by Dunn’s multiple comparisons test. *p < 0.05 **p < 0.01.

Figure 5

Longevity of the chicken intestinal organoids. (a) Brightfield images of chicken intestinal organoids cultured in chicken organoid culture medium. All pictures were taken 2 days after passaging. Scale bar: 400 µm. (b) mRNA levels of LGR5 in 3D embryonic chicken intestinal organoids, expressed as 40-Ct values with GAPDH as a reference gene. Two independent isolations were performed in technical triplicates. The error bars represent the standard deviation of the mean.

Figure 6

Identification of different cell structures in 3D chicken intestinal organoid and the monolayers. Immunohistochemistry images of 3D and 2D structures of chicken intestinal organoids, processed 2 days after seeding. (a,b) Progenitor cells are visualized with anti-Sox9. (c,d) Enteroendocrine cells are visualized by Chromogranin A. (e–g) Tight junctions are visualized with beta-catenin or occludin (h) Mucus-containing goblet cells are visualized using Periodic Acid Schiff (PAS)-positive cell staining of 2D chicken intestinal organoids. The actin filaments are visualized with rhodamine phalloidin (red), and nuclei are stained with DAPI (blue) Scale bar: 400 µm.