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).
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).
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.
The authors were supported by the Polish Ministry of Science and Higher Education grant nos. N N302 130834 to M.P. and N N311 307736 to K.Z. The Faculty of Biochemistry, Biophysics and Biotechnology of the Jagiellonian University, where the microscope images time-lapse recording system was used, is a beneficiary of structural funds from the European Union (grant nos. UDA-POIG.01.03.01-14-036/09-00—“Application of polyisoprenoid derivatives as drug carriers and metabolism regulators” POIG.02.01.00-12-064/08—“Molecular biotechnology for health;” POIG 01.02-00-109/99—“Innovative methods of stem cell applications in medicine”). We would like to acknowledge Professor Tadeusz Tuszynski for providing the access to the microscope facility and Dr. Alan G. Crosby for language correction of the manuscript.
The authors declare no competing interests.
Address correspondence to Malgorzata Pierzchalska, Department of Food Biotechnology, Faculty of Food Technology, University of Agriculture, Balicka 122, 31-149 Kraków, Poland. Email: [email protected]
1.) Kaeffer, B. 2002. Mammalian intestinal epithelial cells in primary culture: a mini-review. In Vitro Cell. Dev. Biol. Anim 38:123-134. 2.) Chopra, D.P., A.A. Dombkowski, P.M. Stemmer, and G.C. Parker. 2010. Intestinal epithelial cells in vitro. Stem Cells Dev 19:131-142. 3.) Sato, T., J.H. van Es, H.J. Snippert, D.E. Stange, R.G. Vries, M. van den Born, N. Barker, N.F. Shroyer. 2011. Paneth cells constitute the niche for Lgr5 stem cells in intestinal crypts. Nature 469:415-418. 4.) Sato, T., R.G. Vries, H.J. Snippert, M. van de Wetering, N. Barker, D.E. Stange, J.H. van Es, A. Abo. 2009. Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature 459:262-265. 5.) Shaker, A., and D.C. Rubin. 2010. Intestinal stem cells and epithelial-mesenchymal interactions in the crypt and stem cell niche. Transl. Res 156:180-187. 6.) Ootani, A., X. Li, E. Sangiorgi, Q.T. Ho, H. Ueno, S. Toda, H. Sugihara, K. Fujimoto. 2009. Sustained in vitro intestinal epithelial culture within a Wnt-dependent stem cell niche. Nat. Med 15:701-706. 7.) Spence, J.R., C.N. Mayhew, S.A. Rankin, M.F. Kuhar, J.E. Vallance, K. Tolle, E.E. Hoskins, V.V. Kalinichenko. 2011. Directed differentiation of human pluripotent stem cells into intestinal tissue in vitro. Nature 470:105-109. 8.) Barker, N., J.H. van Es, J. Kuipers, P. Kujala, M. van den Born, M. Cozijnsen, A. Haegebarth, J. Korving. 2007. Identification of stem cells in small intestine and colon by marker gene Lgr5. Nature 449:1003-1007. 9.) Stephanou, A., D.A. Isenberg, S. Akira, T. Kishimoto, and D.S. Latchman. 1998. The nuclear factor interleukin-6 (NF-IL6) and signal transducer and activator of transcription-3 (STAT-3) signalling pathways co-operate to mediate the activation of the hsp90beta gene by interleukin-6 but have opposite effects on its inducibility by heat shock. Biochem. J 330:189-195. 10.) Messer, M., and A. Dahlqvist. 1966. A one-step ultramicro method for the assay of intestinal disaccharidases. Anal. Biochem 14:376-392. 11.) Blake, D.A., and N.V. McLean. 1989. A colorimetric assay for the measurement of D-glucose consumption by cultured cells. Anal. Biochem 177:156-160. 12.) Uni, Z., A. Geyra, H. Ben-Hur, and D. Sklan. 2000. Small intestinal development in the young chick: crypt formation and enterocyte proliferation and migration. Br. Poult. Sci 41:544-551. 13.) Cohn, S.M., S. Schloemann, T. Tessner, K. Seibert, and W.F. Stenson. 1997. Crypt stem cell survival in the mouse intestinal epithelium is regulated by prostaglandins synthesized through cyclooxygenase-1. J. Clin. Invest 99:1367-1379. 14.) Goessling, W., T.E. North, S. Loewer, A.M. Lord, S. Lee, C.L. Stoick-Cooper, G. Weidinger, M. Puder. 2009. Genetic interaction of PGE2 and Wnt signaling regulates developmental specification of stem cells and regeneration. Cell 136:1136-1147. 15.) Jung, P., T. Sato, A. Merlos-Suárez, F.M. Barriga, M. Iglesias, D. Rossell, H. Auer, M. Gallardo. 2011. Isolation and in vitro expansion of human colonic stem cells. Nat. Med 17:1225-1227. 16.) Nile, C.J., C.L. Townes, G. Michailidis, B.H. Hirst, and J. Hall. 2004. Identification of chicken lysozyme g2 and its expression in the intestine. Cell. Mol. Life Sci 61:2760-2766. 17.) Geyra, A., Z. Uni, O. Gal-Garber, D. Guy, and D. Sklan. 2002. Starving affects CDX gene expression during small intestinal development in the chick. J. Nutr 132:911-917.