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The intestine consists of epithelial cells surrounded by a complex environment as mesenchymal cells and the gut microbiota. With its impressive stem cell regeneration capability, the intestine is able to constantly replenish cells lost through apoptosis or abrasion by food passing through. Over the past decade, researchers have identified signaling pathways involved in stem cell homeostasis such as retinoids pathway. Retinoids are also involved in cell differentiation of healthy and cancer cells. In this study, we describe several approaches in vitro and in vivo to further investigate the effect of retinoids on stem cells, progenitors, and differentiated intestinal cells., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Organoids are 3D ex vivo cell aggregates derived from primary tissue and shown to closely recapitulate tissue homeostasis. Organoids deliver certain advantages compared to 2D cell lines and mouse models, especially in drug-screening studies and translational research projects. The application of organoids in the research field is fast-emerging and new techniques for organoid manipulation are constantly developing. Despite recent advances, RNA-seq-based drug-screening platforms in organoids are not yet established. Here, we provide a detailed protocol for performing TORNADO-seq, a targeted RNA-seq-based drug-screening method in organoids. Analyzing complex phenotypes with a large number of carefully selected read-outs allows to directly classify and group drugs even without structural similarity or overlapping mode of actions from prior knowledge. Our assay principle combines cost-effectiveness and sensitive detection of multiple cell identities, signaling pathways, and key drivers of cellular phenotypes and can be applied to many systems where this new form of high-content screening can provide information not obtainable otherwise., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Intestinal stem cells are responsible for tissue renewal. The study of stem cell properties has become a major challenge in the field. We describe here a method based on Cre recombinase inducible lentivirus vectors that permits delivery of transgenes, either for overexpression or knockdown, in primary stem cells that can be cultured in an 3D intestinal organoid system. This method is an excellent approach for genetic manipulation and can complement in vivo transgenic experiments.