From Pluripotency to Definitive Endoderm: Delineating the Signalling Events of Endoderm Specification in Human Pluripotent Stem Cells

In order to faithfully reproduce human tissues and cell types using pluripotent stem cells, it is essential to have an accurate understanding of the developmental pathways controlling cell fate from early development to mature, specialized cell types. Definitive Endoderm (DE) is an early embryonic cell layer that gives rise to multiple organs such as the gut, liver, pancreas, bladder, thyroid gland, thymus, and lungs. Our group has developed a protocol that efficiently generates hepatocyte like cells (HLCs) from human pluripotent stem cells (hPSCs). This protocol uses the small molecule CHIR99021, a potent activator of canonical WNT signalling to direct hPSCs towards endodermal differentiation. During the differentiation process the predicted sequence of developmental markers was observed, demonstrating a transition through primitive streak to DE using gene expression analysis. This platform allows us to study the cell signalling events that initiate the shift from hPSCs to DE. A detailed understanding of these events will provide the tools to develop more accurate models of human tissues, such as the liver. The signalling events leading to endodermal development involve a myriad of signalling proteins belonging to families such as WNTs, TGFβs, FGFs, BMPs, and the PI3K/Akt pathway. Our results demonstrate new facets of a Wnt-driven interplay between these pathways, uncovering novel relationships between the signalling proteins that drive endodermal differentiation. We demonstrate that the Wnt-mimicking process of GSK3 inhibition, drives DE differentiation, mediating crosstalk between pathways previously established to be important for DE development but not known to be regulated or initiated by canonical Wnt-signalling specifically, such as the PI3K/Akt and Nodal signalling pathways. Furthermore we highlight an interesting pattern of Axin2 and TNKS signalling in DE differentiation. We also show that these events are conserved across different hPSC lines. We therefore conclude that canonical Wnt/GSK3 signalling acts as a master-regulator and initiator of mesendodermal differentiation.