Islet versus enterochromaffin lineage allocation of hPSC-derived pancreatic progenitors can be modulated at both pre-progenitor and post-progenitor stages

Little is known about the molecular mechanisms that underlie the allocation of human pluripotent stem cell-derived pancreatic progenitors (hPSC-PPs) into specific endocrine lineages. By systematically interrogating the components of pancreatic differentiation media, we identify methods to reliably generate hPSC-PPs with high-grade NKX6-1 expression, but find that this alone has a minimal effect on modulating the endocrine composition of hPSC-derived islets. We then test the effect of altering the signaling cues provided after progenitor specification and find that the BMP and FGF/MEK pathways can be manipulated to shift hPSC-PPs between islet and non-islet endocrine lineages. We further find that the method of hPSC-PP specification integrates with these later cues in a way that can be leveraged to selectively increase the frequencies of specific islet lineages or enterochromaffin (EC)-like cells. Using a model of disrupted murine islet development that gives rise to pancreatic EC-like cells, we identify prolonged NEUROG3 expression as a conserved feature associated with both human and murine pancreatic EC-like cell differentiation. In addition to expanding our understanding of pancreatic endocrine lineage allocation, these findings provide some of the first tools to fine-tune composition of hPSC-derived islets for research and therapeutic applications. Overall design: Day 13 human pluripotent stem cell-derived pancreatic progenitors with enhanced NKX6-1 expression were differentiated towards either islet or EC lineages. At day 26, cells were dissociated, viability-sorted, and fixed. 24 hours later, they were sent for fixed RNA profiling.