Human pluripotent stem cell-derived pancreatic ductal organoids to model familial pancreatic cancer in the context of CDKN2a or BRCA2 deficiency

Personalized in vitro models for dysplasia and carcinogenesis in the pancreas have been constrained by insufficient engineering approaches to generate human exocrine pancreatic tissue. Human pluripotent stem cells (hPSCs) may provide the appropriate platform for biomedical studies due to their capability to differentiate into every cell type in the human body. We established a differentiation matrix to generate virtually pure pancreatic duct-like organoids (PDLOs) from hPSCs. These PDLOs showed morphological, transcriptional and functional characteristics of human pancreatic ducts.

Afterwards, we generated an early in vitro cancer model by introduction of a Doxycycline-inducible piggyBac system in hPSCs which allows timed expression of oncogenic KRAS^G12D. Wildtype hPSCs as well as a CRISPR/Cas-engineered CDKN2A^KO/KO line both armed with oncogenic KRAS were differentiated according to our protocol to the ductal lineage, followed by oncogene activation in vitro and in vivo. These PDLOs showed a characteristic growth phenotype of lumen-filling after KRAS^G12D induction, which was further characterized on the molecular level and demonstrated senescence and growth arrest accompanied with increased epithelia-to-mesenchymal transition (EMT). These oncogene-induced responses were even more pronounced in PDLOs with mutated KRAS and genetic covariance of CDKN2A^KO/KO. Orthotopic transplantation was additionally incorporated into the experimental workflow. While transplanted PDLOs with oncogenic KRAS alone developed to well-differentiated tumors, PDLOs with KRAS and CDKN2A-loss resulted in dedifferentiated human pancreatic ductal adenocarcinomas (PDAC) in vivo.

Summarized, our novel and unique differentiation platform generates human untransformed ducts and allows modelling of plasticity, dysplasia, and cancer formation in a human and genetically defined background in the pancreas. Thereby, we also show that human ducts are indeed permissive to generate PDAC. Tailored genome editing strategies mimicking the mutational make-up of human PDAC will open novel opportunities to provide a unique and valuable human PDAC model.

Publication History

Article published online:
08 September 2020