Single-cell-resolved differentiation of human induced pluripotent stem cells into pancreatic duct-like organoids on a microwell chip

Creating in vitro models of diseases of the pancreatic ductal compartment requires a comprehensive understanding of the developmental trajectories of pancreas-specific cell types. Here we report the single-cell characterization of the differentiation of pancreatic duct-like organoids (PDLOs) from human induced pluripotent stem cells (hiPSCs) on a microwell chip that facilitates the uniform aggregation and chemical induction of hiPSC-derived pancreatic progenitors. Using time-resolved single-cell transcriptional profiling and immunofluorescence imaging of the forming PDLOs, we identified differentiation routes from pancreatic progenitors through ductal intermediates to two types of mature duct-like cells and a few non-ductal cell types. PDLO subpopulations expressed either mucins or the cystic fibrosis transmembrane conductance regulator, and resembled human adult duct cells. We also used the chip to uncover ductal markers relevant to pancreatic carcinogenesis, and to establish PDLO co-cultures with stellate cells, which allowed for the study of epithelial–mesenchymal signalling. The PDLO microsystem could be used to establish patient-specific pancreatic duct models. A microwell chip facilitates the single-cell characterization of the differentiation of aggregates of human induced pluripotent stem cells into pancreatic duct-like organoids and the discovery of secreted markers of pancreatic carcinogenesis.