AKT/GSK3β signaling pathway is critically involved in human pluripotent stem cell survival

LEONARDO ROMORINI; XIMENA GARATE; GABRIEL NEIMAN; CARLOS LUZZANI; VERÓNICA FURMENTO; ALEJANDRA GUBERMAN; GUSTAVO SEVLEVER; MARÍA ELIDA SCASSA; SANTIAGO G. MIRIUKA

San Francisco

Congreso; ISSCR Annual Meeting; 2016

ISSCR

Human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) are self-renewing pluripotent stem cells (PSC) that can differentiate into a wide range of specialized cells. Basic fibroblast growth factor(bFGF) has been proved to be essential to both hESCs and hiPSCs survival, stemness and self-renewal. One of itsdownstream targets, PI3K, and its most prominent effector, AKT, regulate cell viability and apoptosis in many different cell types. Although it is well known that PI3K/AKT activation by bFGF is relevant for PSC stemness maintenance,the role of this signaling pathway on PSC survival has not yet been fully elucidated. The aim of this work wasto explore the relevance and molecular mechanisms involved in the regulation of hESCs (WA09/H9 and WA01/H1 lines) and hiPSCs (FN2.1 line) survival by AKT. We found that pharmacological inhibition of AKT with threenon-structurally related inhibitors (GSK690693, AKT inhibitor VIII and AKT inhibitor IV) decreased cell viabilityin a concentration dependent manner. We next analyzed several aspects of programmed cell death and found anincrease in the translocation of phosphatidylserine (PS) to the outer leaflet of the plasma membrane and in the extentof DNA fragmentation as soon as 8 hours after AKT inhibitors addition in PSC. Moreover, Western blot analysisrevealed the activation of the initiator Caspase-9, the effector Caspase-3, and PARP cleavage at different timepoints after AKT inhibition. However, no relevant changes in BCL-2 family members BCL-XL, BCL-2 and BAX proteinproducts were found. Importantly, we observed that GSK3β signaling is responsible, at least in part, of the apoptosisinduction triggered by AKT inhibition. Importantly, pharmacological inhibition of GSK3β with CHIR99021 decreasedbasal apoptosis rates and induced proliferation of PSC cultured under standard conditions. Collectively, inthis study we demonstrated that AKT signaling activation, partly mediated by GSK3β inhibition, prevents apoptosisand thus results relevant for human PSC survival.