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Organoids, a multi-cellular and organ-like structure cultured in vitro , can be used in a variety of fields such as disease modeling, drug discovery, or cell therapy development. When organoids derived from Lgr5 stem cells are cultured ex vivo , recombinant R-spondin-1 protein should be added at a high concentration for the initiation and maintenance of the organoids. Because the addition of large amounts of R-spondin-1 greatly increases the cost of organoids, the organoids grown with R-spondin-1 are not practical for large-scale drug screening and for the development of therapeutic agents. In this study, we tried to find a R-spondin-1 substitute compound that is able initiate small intestinal organoids without the use of the R-spondin-1 protein; thus, using organoid media that each included one compound from among an 8,364 compound library instead of R-spondin-1, we observed whether organoids were established from the crypts of the small intestine. As a result, we found one compound that could promote the initial formation and growth of enteroids in the medium without R-spondin-1 and named it RS-246204. The enteroids grown with RS-246204 had a similar differentiation capacity as well as self-renewal capacity as the enteroids grown with R-spondin-1. Furthermore, the RS-246204-derived enteroids could successfully produce the forskolin induced swelling and the organoid based epithelial to mesenchymal transition model. This compound could be used for developing a cost-efficient culturing method for intestinal organoids as well as for exploring Lgr5 signaling, intestinal stem cell physiology and therapeutics for GI tract diseases.

Effects of a small molecule R-spondin-1 substitute RS-246204 on a mouse intestinal organoid culture