Directed differentiation of human pluripotent stem cells into pancreatobiliary co-progenitor-like population

Progress in uncovering the causes of extrahepatic biliary diseases and developing new therapies has been constrained by the inaccessibility of donor tissue and a lack of experimental models. Although hepatic, intrahepatic biliary, and pancreatic 2D/3D models have been successfully established from pluripotent stem cells (PSCs), in vitro generation of extrahepatic biliary cells remains a major challenge, due to the absence of developmental cues. Here we report a de novo method for directed differentiation of human PSCs (both embryonic and induced) into pancreato-biliary progenitors-like cells (PBPLCs). We showed that temporal manipulation of the FGF, Vc, WNT, BMP and retinoic acid signaling enabled generation of 3D PBPLCs spheroids manifesting PDX1⁺ SOX17⁺ TBX3^- phenotype. These PBPLCs possess bi-potential that could be further directed to Insulin⁺ NKX6.1⁺ pancreatic islet β-like cells, or differentiated into gallbladder-like organoids in vitro, respectively, via addition of their developmentally relevant signaling molecules. Formed gallbladder-like organoids are comprised of both epithelial and mesenchymal compartments, self-organized with structural features similar to the native gallbladder. The epithelia in organoids expressed many proteins and genes that highly enriched in extrahepatic cholangiocytes, including SOX17, CLDN3, DBA, CK7, MUC5B, FGF19, CHST4., etc. Our method for generation and purification of PSC-derived PBPLCs opens a new avenue for study of biological mechanisms controlling pancreato-biliary development, as well as their disease modeling and drug screening.