Organoid culture and 3D bioprinting of pluripotent stem cell-derived pancreatic progenitors

In the mammalian embryo, the developing pancreas is surrounded by mesoderm compartments, which are important for generating the signals and mechanical constraints that induce the formation of the pancreatic bud and its differentiation. Most protocols for pancreatic organoids do not include this mesoderm niche and have partial success in capturing the full pancreas repertoire. This work aims to generate a tissue-like embryonic pancreas using either organoid culture or three-dimension (3D) bioprinting, without the use of poorly defined extracellular matrix such as Matrigel. The first step in our work focused on mouse embryonic stem cells (mESCs) differentiation in adherent culture. We were able to achieve a higher differentiation efficacy to definitive endoderm and specifically to pancreatic progenitors (PPs) by first differentiating mESCs to epiblast stem cells (EpiSCs), a primed pluripotent state. Next, we aggregated PPs with EpiSC-derived mesoderm progenitors (MPs), recapitulating the natural pancreas development, which is affected by adjacent mesoderm compartments in the embryo. In the bioprinting technique we incorporated PPs with endothelial and mesenchymal cells due to their importance for functional maturation in vitro. In the organoid culture we utilize the cells’ self-organization, whereas the 3D bioprinter is used to print cells in specified locations.

Both culture techniques recapitulated the different pancreatic cell types and contained endocrine, acinar and ductal cells, together with blood vessel-like networks. Furthermore, analysis of single-cell RNA sequencing of the organoid culture revealed expression patterns similar to those of the mouse embryonic pancreas at E12, E14 and E17, primarily in the endocrine differentiation lineage.