Analysis of Differentiation Protocols Defines a Common Pancreatic Progenitor Molecular Signature and Guides Refinement of Endocrine Differentiation

Several distinct differentiation protocols for deriving pancreatic progenitors (PPs) from human pluripotent stem cells have been described, but it remains to be shown how similar the PPs are across protocols and how well they resemble their in vivo counterparts. Here, we evaluated three differentiation protocols, performed RNA and assay for transposase-accessible chromatin using sequencing on isolated PPs derived with these, and compared them with fetal human pancreas populations. This enabled us to define a shared transcriptional and epigenomic signature of the PPs, including several genes not previously implicated in pancreas development. Furthermore, we identified a significant and previously unappreciated cross-protocol variation of the PPs through multi-omics analysis and demonstrate how such information can be applied to refine differentiation protocols for derivation of insulin-producing beta-like cells. Together, our study highlights the importance of a detailed characterization of defined cell populations derived from distinct differentiation protocols and provides a valuable resource for exploring human pancreatic development. •Systematic comparison of three protocols for hPSC-pancreatic progenitor differentiation•Individual hPSC lines differentiate preferentially with different protocols•Shared transcriptomic and epigenomic signatures of hPSC-pancreatic progenitors•Molecular characterization of hPSC-pancreatic progenitors guides protocol refinement Wesolowska-Andersen and colleagues present a comprehensive molecular and functional analysis of human PSC-derived pancreatic progenitors derived using three differentiation protocols. They define their shared transcriptomic and epigenomic signatures but also highlight significant differences between protocols, which are then used to guide protocol modifications to enhance further differentiation toward endocrine lineage.