Akt/gsk3b signaling pathway is critically involved in human pluripotent stem cell survival

ROMORINI L; LUZZANI C; SEVLEVER G; NEIMAN G; GUBERMAN A; GARATE X; FURMENTO V; MIRIUKA S

San Francisco

Congreso; ISSCR annual meeting 2016; 2016

Human embryonic stem cells (hESCs) and human inducedpluripotent stem cells (hiPSCs) are self-renewing pluripotentstem cells (PSC) that can differentiate into a widerange of specialized cells. Basic fibroblast growth factor(bFGF) has been proved to be essential to both hESCsand hiPSCs survival, stemness and self-renewal. One of itsdownstream targets, PI3K, and its most prominent effector,AKT, regulate cell viability and apoptosis in many differentcell types. Although it is well known that PI3K/AKTactivation by bFGF is relevant for PSC stemness maintenance,the role of this signaling pathway on PSC survivalhas not yet been fully elucidated. The aim of this work wasto explore the relevance and molecular mechanisms involvedin the regulation of hESCs (WA09/H9 and WA01/H1 lines) and hiPSCs (FN2.1 line) survival by AKT. Wefound that pharmacological inhibition of AKT with threenon-structurally related inhibitors (GSK690693, AKT inhibitorVIII and AKT inhibitor IV) decreased cell viabilityin a concentration dependent manner. We next analyzedseveral aspects of programmed cell death and found anincrease in the translocation of phosphatidylserine (PS) tothe outer leaflet of the plasma membrane and in the extentof DNA fragmentation as soon as 8 hours after AKTinhibitors addition in PSC. Moreover, Western blot analysisrevealed the activation of the initiator Caspase-9, theeffector Caspase-3, and PARP cleavage at different timepoints after AKT inhibition. However, no relevant changesin BCL-2 family members BCL-XL, BCL-2 and BAX proteinproducts were found. Importantly, we observed thatGSK3β 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 ofPSC 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