OPTIMIZING DIFFERENTIATION AND REGIONAL PATTERNING OF GASTROINTESTINAL ORGANOIDS FROM PLURIPOTENT STEM CELLS FOR STUDYING INFLAMMATORY BOWEL DISEASE

Abstract BACKGROUND Recent advances in gastrointestinal (GI) organoids from PSCs have enabled modeling crosstalk among epithelial, mesenchymal, and immune cells. Moreover, by modulating Wnt, FGF, and BMP signals, GI organoids can be regionalized into Human Intestinal Organoids (HIO) with duodenal (duo-HIO) or ileal (ile-HIO) identity, or Human Colonic Organoids (HCO). These traits make them a key platform for studying the transmural and multiregional pathophysiology of Crohn’s disease (CD). Given experimental variability in the differentiation ability into different GI regions, as well as across different PSC lines, we aimed to optimize current HIO/HCO protocols by analyzing each differentiation step using multiple PSC lines. METHODS This study used iPSC lines from CD patients and hESC. PSCs were treated with Activin A for 3 days, followed by FGF4 and CHIR99021 for 4 days (duo-HIO/HCO) or 6 days (ile-HIO). Mid-hindgut (MHG) spheroids were cultured with EGF to induce duo/ile-HIO. For HCO, BMP2 was added for the first 3 days. Cells and organoids were analyzed by immunostaining and qPCR. Immunostaining with PDX1, CDX2, SATB2 and GATA3 confirmed duodenal, intestinal, ileal/colon, and urothelial identity, respectively. RESULTS Using the original protocols, we and others observed regional heterogeneity in organoids at the end of differentiation, approximately 4 weeks in culture. In duo-HIO conditions we observed organoids with duodenal and non-duodenal intestinal domains. In ile-HIO/HCO conditions, we observed co-emerging urothelium. These results suggest that changes in reagents since the original protocols were established is causing regional drift. We hypothesized that activities of Activin A and FGF4 may be higher. By optimizing these we observed more robust and reproducible production of uniformly regionalized organoids. For example, more uniform duo-HIOs can be generated by lowering FGF4. The second significant area of variability was in spheroid viability across iPSC lines and experiments. We observed that spheroids were less viable when the ratio of FOXF1+ mesodermal to CDX2+ cells was too low at the MHG stage. The main drivers of this ratio were the activities of Activin A and FGF4. Compared to the original protocols, we found that the recommended concentrations of Activin A and FGF4 were too high and negatively impacted mesoderm. By lowering both and adding ROCK inhibitor for the first 3 days of spheroid culture, we found that the growth of spheroids and production of HIOs and HCOs from even difficult iPSC lines were vastly improved. CONCLUSIONS Through methodical analysis of each stage of HIO/HCO differentiation across multiple PSC lines, we identified that changes in reagent activities over the years may be the driver of variability in GI organoid production. Our current optimized protocol yields a higher number of properly regionalized organoids.