High-content screening of organoids reveals the mechanisms of human pancreas acinar specification

Organoids derived from pluripotent stem cells have emerged as powerful models to study human development. To investigate signaling pathways regulating human pancreas differentiation and morphogenesis, we developed a high-content, image-based screen and quantitative multivariate analysis pipelines robust to heterogeneity to extract single-cell and organoid features using pancreatic progenitor organoids. Here, we identified 54 compounds affecting cell identity and/or morphological landscape. Focusing on one family of compounds, we found that glycogen synthase kinase 3α/β (GSK3A/B) inhibition via wingless/int-1 (WNT) signaling has a reversible effect on cell identity, repressing pancreatic progenitor markers and inducing a poised state in progenitors transitioning to acinar cells. We show that additional fibroblast growth factor (FGF) repression enables further differentiation of acinar cells, recapitulating pancreatic acinar morphogenesis and function. The ability to produce acinar cells is valuable for future studies on pancreatic exocrine function and cancer initiation in humans, as acinar cells are thought to be an important cell of origin for pancreatic adenocarcinoma. • High-content screen reveals 54 compounds altering organoid shape or differentiation • Analysis methods robust to organoid heterogeneity are established • Screen-derived GSK3A/B inhibitors control pancreatic acinar cell differentiation Keshara et al. conducted a high-content screen on pancreas organoids that identified 54 compounds affecting differentiation or morphogenesis. After validating 11 compounds, they focus on Gsk3A/B inhibitors, which, combined with FGF withdrawal, drive acinar cell differentiation, enabling future studies on these cells and their role in cancer initiation in humans.