Protocol for in vitro modeling of specification and morphogenesis of early pancreas development using human pluripotent stem cell-based organoid differentiation

Here, we present a protocol to generate key pancreatic cell types in vitro using human pluripotent stem cell (hPSC)-based Matrigel-overlay organoid differentiation. These include multipotent and bipotent progenitors, endocrine progenitors, and hormone-producing endocrine cells. We describe steps for culturing hPSCs as a 2D monolayer, applying a Matrigel overlay to create a 3D epithelial niche, and guiding stepwise differentiation. This system supports live-cell imaging and real-time tracking of morphogenesis and fate decisions, providing a platform for studying organ development and disease. For complete details on the use and execution of this protocol, please refer to Ulf et al.¹.