GRP94 is indispensable for definitive endoderm specification of human induced pluripotent stem cells

Human induced pluripotent stem cell (hiPSC)-derived insulin-producing β cell therapy shows promise in treating type 1 diabetes and potentially type 2 diabetes. Understanding the genetic factors controlling hiPSC differentiation could optimize this therapy. In this study, we investigated the role of glucose-regulated protein 94 (GRP94) in human β cell development by generating HSP90B1/GRP94 knockout (KO) hiPSCs, re-expressing GRP94 in the mutants and inducing their β cell differentiation. Our results revealed that GRP94 depletion hindered β cell generation by promoting cell death induced by endoplasmic reticulum (ER) stress and other stressors during definitive endoderm (DE) differentiation. Moreover, GRP94 deletion resulted in decreased activation of WNT/β-catenin signaling, which is critical for DE specification. Re-expression of GRP94 in GRP94 KO iPSCs partially reversed DE differentiation deficiency and alleviated cell death. These findings highlight the previously unrecognized indispensable role of GRP94 in human DE formation and consequent β cell development from hiPSCs. GRP94 mitigates ER stress-induced cell death and regulates the WNT/β-catenin signaling pathway, which is both crucial for successful β cell differentiation. These results provide new insights into the molecular mechanisms underlying β cell differentiation from hiPSCs and suggest that targeting GRP94 pathways could enhance the efficiency of hiPSC-derived insulin-producing cell therapies for diabetes treatment.