Inhibition of Proteasome Activity Facilitates Definitive Endodermal Specification of pluripotent Stem Cells by influencing YAP signaling

Understanding the molecular players that control the specification of definitive endoderm is imperative to obtain the homogenous population of pancreatic β-cells from stem cells. Though the Ubiquitin proteasome system (UPS) has been envisaged as a crucial intracellular protein degradation system, its role in germ layer specification remains elusive. In this study, using a mouse embryonic stem cells model system (mESCs) we observed decreased proteasomal activity specifically during endoderm, but not in meso- or ecto-derm differentiation. Extraneous inhibition of proteasomal activity during differentiation enhanced the expression of endodermal genes specifically. Enhancing proteasomal activity by including the activator IU1 in the induction culture, inhibited definitive endodermal differentiation. Further, inhibiting proteasomal activity at the definitive endodermal stage resulted in enhanced generation of insulin-positive cells. A similar increase in endodermal gene expression by inhibiting proteasomal activity was observed in miPSC and hiPSC differentiated towards endodermal lineage. Mechanistic insight showed no contribution of endoplasmic reticulum unfolded protein response but revealed the involvement of the YAP signaling pathway in proteasome-inhibited enhanced endodermal differentiation. Unravelling the specific involvement of UPS in endodermal cell fate specification in pluripotent stem cells paves the way for obtaining better qualitative and quantitative definitive endodermal cells for plausible cellular therapy in the future.