AbstractBackgroundInduced pluripotent stem cells (iPSC) can be differentiated to cells in all three germ layers, as well as cells in the extraembryonic tissues. Efforts in iPSC differentiation into pancreatic progenitorsin vitrohave largely been focused on optimizing soluble growth cues in conventional two-dimensional (2D) culture, whereas the impact of three-dimensional (3D) matrix properties on the morphogenesis of iPSC remains elusive.MethodsIn this work, we employ gelatin-based thiol-norbornene photo-click hydrogels forin situ3D differentiation of human iPSCs into pancreatic progenitors (PP). Molecular analysis and single cell RNA-sequencing were utilized to elucidate on the distinct identities of subpopulations within the 2D and 3D differentiated cells.ResultsWe found that, while established soluble cues led to predominately PP cells in 2D culture, differentiation of iPSCs using the same soluble factors led to prominent branching morphogenesis, ductal network formation, and generation of diverse endoderm populations. Through single-cell RNA-sequencing, we found that 3D differentiation resulted in enrichments of pan-endodermal cells and ductal cells. We further noted the emergence of a group of extraembryonic cells in 3D, which was absent in 2D differentiation. The unexpected emergence of extraembryonic cells in 3D was found to be associated with enrichment of Wnt and BMP signaling pathways, which may have contributed to the emergence of diverse cell populations. The expressions of PP signature genes PDX1 and NKX6.1 were restored through inhibition of Wnt signaling at the beginning of the posterior foregut stage.ConclusionsTo our knowledge, this work established the first 3D hydrogel system forin situdifferentiation of human iPSCs into PPs. Ongoing work focuses on enhancing pancreatic differentiation efficiency through modulating physicochemical properties of the iPSC-laden matrices.