Mapping cellular diversity and lineage dynamics within the developing murine and human pancreas

The pancreas is a highly branched, compound gland whose development requires the diversification of many distinct cell lineages. Cells in exocrine compartment of the pancreas synthesize and secrete digestive enzymes required for food digestion, whereas cells in the endocrine compartment are responsible for maintaining glucose homeostasis through the production of various hormones. In this work, we sought to characterize the diversity of cell types present in the developing pancreas and chart their differentiation through multiple developmental stages. We start in Chapter 2 by identifying novel and known cell populations within the mesenchymal and epithelial compartments of the developing murine pancreas and mapping their developmental progress across multiple embryonic stages using single-cell RNA-sequencing. We uncover significant cellular diversity within the mesenchymal compartment and utilize single-cell transcriptomic data to reconstruct lineage relationships among the developing pancreatic mesothelium and several previously uncharacterized mesenchymal populations in mouse pancreatic development. In the epithelial compartment, we uncover a novel endocrine progenitor stage defined by high expression of a transcription factor named Fev. Through genetic lineage tracing, we demonstrate that this Fev+ progenitor population is derived from Ngn3+ endocrine progenitors and that all hormone-expressing endocrine lineages of the murine pancreas transit through a Fev-expressing cell stage. Through in silico reconstruction of endocrine lineages, we identify candidate regulators of alpha and beta lineage allocation expressed during this novel Fev+ progenitor stage. In Chapter 3, we apply our findings of endocrine lineage dynamics in murine pancreatic development to that of human and uncover similar FEV-expressing endocrine progenitor populations in human pancreatic development. We map the transcriptional dynamics of human endocrine cell differentiation and identify novel candidate lineage regulators of human alpha and beta lineage allocation. We subsequently identify major disparities between the developmental paths that human endocrine cells follow in vivo versus in vitro during directed differentiation of hESCs towards the beta lineage. Our analysis reveals a lineage that may be mis-differentiated as hESC-derived progenitors differentiate towards a beta cell fate. Blocking the generation of this mis-differentiated lineage during in vitro beta cell differentiation represents a powerful solution in making in vitro beta cell differentiation more robust. Finally, given that FEV+ progenitors constitute a major stage in human endocrine cell differentiation, we have generated novel tools to study both the function of FEV and FEV-expressing cells in in vitro beta cell differentiation. Our work forms a foundation on which improvements to in vitro beta cell differentiation can be made to more closely reflect proper endocrine cell development in vivo, thereby increasing beta cell yield at the end of this process and generating beta cells that are functional