Abstract
AbstractIn 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 of pancreatic organoid generation do not include this mesoderm niche and have partial success in capturing the full repertoire of the pancreas. This work aims to generate a pancreatic organoid by differentiating mouse embryonic stem cells (mESCs) under controlled conditions to mesoderm in parallel to pancreas identity, without the use of poorly defined extracellular matrix such as Matrigel. The first step in our work focused on mESCs differentiation in adherent culture. We were able to obtain a higher differentiation efficacy to definitive endoderm and specifically to pancreas progenitors (PPs) by first differentiating mESCs to epiblast stem cells (EpiSCs), a primed pluripotent state. Next, we built a three-dimension (3D) culture, by aggregating PPs with EpiSC-derived mesoderm progenitors (MPs), recapitulating cellular interactions occurring during embryonic pancreas development. Culture of these MP-PP organoids, referred here as pancreatic organoids, generated the different pancreatic cell types, i.e. endocrine, acinar and ductal cells, as well as blood vessel-like networks. Furthermore, single-cell RNA sequencing analysis of the pancreatic organoids revealed gene expression patterns similar to those of the mouse embryonic pancreas at E12, E14 and E17, primarily in the endocrine differentiation lineage.
Publisher
Cold Spring Harbor Laboratory