Relevance of akt signaling pathway on human pluripotent stem cells survival.

ROMORINI L; GARATE X; NEIMAN G; LUZZANI C; SCASSA M; SEVLEVER G; GUBERMAN A; MIRIUKA S

Congreso; I Latin American VII Brasilian and I Argentine Congress of Stem Cells and Cell Therapy; 2014

INTRODUCTION: Human embryonic and induced pluripotent stem cells (hESCs and hiPs, respectively) have great capacity to differentiate in vitro and huge potential to be usedas cellular sources for regenerative medicine. The basic fibroblast growth factor (bFGF) is essential for survival, maintenanceof pluripotency and self-renewal of hESCs and hiPs. PI3K and its most prominent effector, Akt, control cell viability in many cell types. bFGFactivates PI3K signaling pathway in these pluripotent cells. OBJETIVES: The aim of this work is to studythe contribution of Akt in hESCs? and hiPs? viability. METHODS: hESCs lines WA01 (H1) - WA09 (H9) and hiPs line FN2.1 (generated in our laboratory from human foreskin fibroblasts) were cultured until confluence in the presence of bFGF. Then, cell survival was analyzed in the presence of three structurally unrelated Akt inhibitors. Inhibitors used were: Akt inhibitor IV (specific of a kinase which activates Akt), Akt inhibitor VIII (prevents bindingof Akt to cell membrane) and GSK690693 (blocksATP-binding site of Akt). In particular, cell viability was measured by the XTT colorimetric assay and Trypan blue exclusion test. Annexin V exposure to outer cell membrane and DNA fragmentation are two of the most important criteria used to indentify apoptotic cells, therefore changes in apoptotic rate were analyzed assessing DNA fragmentation with a Cell Death Detection Elisa Kit and determining Annexin V/propidium iodide double staining with a flow cytometer. Activation of initiator and effector caspases (like caspase-9 and caspase-3, respectively) is another hallmark of apoptosis induction. In that sense, caspase-9, caspase-3 and PARP (caspase-3 substrate) cleavage were measured by Western blot. Besides, changes in Bcl-2 family members abundance were also quantified by Western blot. RESULTS: The pluripotent nature of confluent hESCs and hiPs was confirmed by the presence of the pluripotent markers: TRA1-60, TRA1-81 SSEA-4, Oct-4 and Nanog in immunofluorescence and quantitative RT-PCR assays. Cell viability significantly decreased in a concentration dependent manner in the presence of each inhibitor. Moreover, in all cases, the percentage of viable cells also diminished upon Akt inhibition. Furthermore, an increase in the number of Annexin V+/propidium iodide- cells and in the extent of DNA fragmentation were observed after 8 hours of Akt inhibitors addition inH9, H1 and FN2.1 cells. Moreover, Western blot analysis revealed the activation of caspase-9, caspase-3 and PARP cleavage at different time points upon Akt inhibitors treatment. Finally, no changes in Bcl-2 family members expression levels were detected by Western Blot under the same experimental conditions. CONCLUSIONS: Taken together, we conclude from the above results, that AKT activity is anti-apoptotic and thus relevantto hESCs and hiPs survival.