Cohn and collaborators previously reported that PGE₂ promotes stem cell growth and survival after intestine radiation-induced injury (13). It was also suggested that the interaction of Wnt and PGE₂ pathways is a master regulator of vertebrate hematopoietic stem cell proliferation during regeneration and recovery (14). PGE₂ was already used to stimulate the growth of organoids in the case of human colonic stem cell culture as an additive to a medium containing Wnt3a, R-spondin 1, Noggin, gastrin, and vitamin nicotinamide (15). Therefore, we set out to determine whether PGE₂ was able to substitute for R-spondin 1 and Noggin in organoid supporting medium. It emerged that this prostaglandin is very effective in sustaining survival and promoting growth of epithelial spheres in a Matrigel matrix (Figure 2A and Figure 3). Long-term culture confirmed that epithelial spheroids were still viable and visible in Matrigel after 5 weeks (Figure 2D). Immunoblot analysis proved that chicken organoids comprise cells expressing epithelial intermediate filaments (cytokeratin) and markers of differentiating and differentiated enterocytes (villin). The expression level of these proteins in organoids remains stable regardless of cell culture conditions (Figure 2B). The presence of Sox-9, a stem/progenitor population marker and member of the Sry-box transcription factor family, was detected by immunoprecipitation followed by immunoblotting. In mammals, Sox-9 is expressed at the bottom of the crypts both in stem cells and in Paneth cells; however, Paneth cells are thought not to exist in bird intestine (16). Therefore, we anticipated that the amount of Sox-9 protein in chicken tissue would be proportional to the number of intestinal stem cells. In organoids grown in the presence of PGE₂, Sox-9 protein is as abundant as in the R-spondin 1- and Noggin-treated cells. In cultures treated with EGF alone, Sox-9 protein expression was dramatically lower (Figure 2B).

Figure 3. Organoids were cultured for 4 days, and bright field images were captured with a digital camera. (Click to enlarge)

Similar to mammals, the expression of caudal family transcription factors (cdx) in birds is restricted to the intestine of adult animals, and these genes are presumed to be responsible for activating a differentiation program in enterocyte progenitors. A study by Geyra et al. concluded that chicken cdxA gene is a homolog of mammals’ cdx2 and is preferentially expressed in the epithelium of the villi. The chicken cdxB expression pattern indicates that it is present in all intestinal epithelial cells (17). Semiquantitative RT-PCR showed that both cdxA and cdxB are expressed in cultured organoids. Organoids cultured in the presence of R-spondin 1 and Noggin or PGE₂ are characterized by a higher CdxA mRNA level. This indicates that in those conditions, SOX-9 positive cells exist in the proliferative compartment as well as on the intestinal differentiation pathway (Figure 4). Sucrase activity measurements show that organoids cultured in the presence of Wnt-agonists or PGE₂ express high levels of this brush border hydrolase activity (not shown), indicating the presence of mature enterocytes. Cells cultured for 1 week in the presence of PGE₂ demonstrated the activity of 4.1 mU/mg protein ± 2.5 mU/mg, and in the presence of Wnt-agonists, cells demonstrated the activity of 2.1 mU/mg protein ± 2.5 mU/mg (mean ± sd, n = 3, the difference is not significant).

Figure 4. Transcript abundance of cdxA and cdxB genes expressed in cells building organoids kept in various culture conditions as compared with ß-actin. (Click to enlarge)

The high cost of media required to culture intestinal organoids hampers use of the model in large-scale industrial and laboratory applications. Therefore PGE₂ is an attractive and much less expensive alternative to the recombinant Wnt agonists. The effectiveness of this method remains to be investigated in mammalian models. However, our method of chicken organoid culture could be potentially used as a source for differentiated epithelial cells. This opens up new opportunities for studies on avian intestine physiology, investigations of the mechanisms of drugs and feed absorption, and research on gut immunity.