Photo-click Decellularized Matrix Hydrogels for Generating Pancreatic Ductal Organoids

Abstract Pancreatic ductal organoids (PDOs) generated from human induced pluripotent stem cells (iPSCs) can be used to model pancreatic diseases and to conduct drug screening/testing. However, current protocols for generating PDOs rely heavily on tumor-derived Matrigel, which has been shown to upregulate oncogenes. Furthermore, Matrigel’s undefined composition and weak mechanical properties hamper mechanistic studies of cell-material interactions. In this study, we explore photo-clickable decellularized small intestine submucosa-norbornene (dSIS-NB) hydrogels as a Matrigel replacement for generating human iPSC-derived PDOs. To achieve this, pancreatic progenitors (PP) were first differentiated in conventional two-dimensional (2D) culture. PP cells were aggregated into spheroids, then encapsulated and differentiated within dSIS-NB hydrogels with tunable stiffness. The differentiated organoids were analyzed by morphology, expression of key pancreatic ductal markers, and single-cell RNA sequencing (scRNA-seq). PDOs generated in stiffer photo-clickable dSIS-NB hydrogels (shear moduli ~2.5 kPa) underwent pronounced mesenchymal-epithelial transition (MET) during differentiation and maintained ductal epithelial phenotype post-differentiation. In contrast, differentiation of PP spheroids in softer dSIS-NB gels (shear moduli ~0.9 kPa) and Matrigel resulted in a persistent mesenchymal phenotype and failed to generate functional PDOs. Finally, scRNA-seq results revealed that stiffer dSIS-NB hydrogels strongly biased ductal cell differentiation, achieving >97% ductal induction efficiency.

Keywords: Pancreatic ductal organoids, induced pluripotent stem cells, hydrogels, matrix stiffness, single-cell RNA sequencing, mesenchymal-epithelial transition

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