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

Abstract

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.

Organotypic and Microphysiological Human Tissue Models for Drug Discovery and Development—Current State-of-the-Art and Future Perspectives

“…To support the development of intestinal organoids, medium is typically supplemented with EGF, Noggin, Wnt3a, and R-spondin (Holmberg et al, 2017). However, small molecule-based approaches that replace Wnt-3a with Chir99021 (Langlands et al, 2018;, Noggin with LDN-193189 (Liu et al, 2018b), and R-spondin with RS-246204 (Nam et al, 2017) have been presented. Single cells first grow into 3D Human Tissue Models cyst-like aggregates, which upon prolonged culture assume a budding-like morphology.…”

The application of intestinal organoids and their co-culture systems in the study of gastrointestinal diseases

“…In addition, neuregulin 1 (NRG1), a key EGF family ligand produced by mesenchymal stromal cells, macrophages, and Paneth cells, is important for the proliferation of intestinal crypts and organoid formation, as well as for regeneration after injury [29]. A recent study also demonstrated that R-spondin 1 could be substituted by a compound named RS-246204 for mouse intestinal organoid culture [30]. Moreover, the modulation of Wnt and Notch signaling in intestinal organoids can regulate IEC lineage differentiation [31].…”

3D organotypic cell structures for drug development and Microorganism-Host interaction research

“…It was demonstrated that the addition of DAPT or BMP molecules to the culture medium is sufficient to stimulate the differentiation of ISCs and the generation of multicellular intestinal organoids. Moreover, some studies have been carried out to search for methods to reduce the cost of culture media, which reported that the Noggin protein can be replaced by LDN-193189, and R-spondin 1 protein can be substituted by RS-246204 (Liu et al 2018;Nam et al 2018;Rahmani et al 2019).…”