Generation of an optimized model of iPSC-derived human intestinal organoids for the expression of an intestinal stem cell fluorescent reporter, and a preliminary assessment for its application in high-throughput screening

The study of human diseases requires the development of modelling systems that accurately replicate both the normal physiology and the progression of the disease. However, commonly used cell culture models are unable to replicate key features of the tissue microenvironment such as the three-dimensional microarchitecture, organization, cellular diversity, and distribution. Organoid technology has overcome these limitations, generating better and more accurate models. The aim of this work was to generate a model of human intestinal organoids (HIOs) derived from induced pluripotent stem cells (iPSCs), expressing an LGR5-GFP reporter within the stem cell population, and implement it in high-throughput experiments for drug discovery studies. We have successfully generated an optimized protocol for the generation of HIOs from iPSCs. The organoids were comprised by an inner layer of columnar intestinal epithelium supported by an outer layer of mesenchymal cells. The gene expression profile revealed a differentiation bias towards the absorptive lineage. Our optimized protocol reduced the time and cost required for the production of the organoids, favouring its compatibility with high-throughput experiments. Excitingly, this protocol was implemented for the generation of intestinal epithelium tubules on an organ-on-a-chip format from in situ differentiated iPSC. Next, we attempted to generate an LGR5-GFP reporter iPS cell line to track the ISCs within the organoids; however, this task proved unsuccessful, and further comparisons with the literature suggested an inherent issue with the detection of human LGR5 using fluorescence-based techniques. Finally, in light of the results obtained from the LGR5-GFP reporter, and other technical limitations, we performed a preliminary high-throughput small-molecule screening on FUCCI-CaCo-2 cells, instead of LGR-GFP HIOs. The results of the screening identified a subset of compounds with an effect over the G1 phase of the cell cycle. Given the role of G1 in the self-renewal, differentiation, and malignant transformation of the ISCs, these compounds could potentially serve as regulators of the activity of the ISC population. Future work will focus on the validation of these results in HIOs.