Relevance of AKT signaling pathway on human pluripotent stem cell survival

LEONARDO ROMORINI; XIMENA GARATE; GABRIEL NEIMAN; CARLOS LUZZANI; MARIA ELIDA SCASSA; GUSTAVO SEVLEVER; ALEJANDRA S. GUBERMAN; SANTIAGO G. MIRIUKA

Congreso; I Latin American - VIII Brazilian and I Argentine Congress of Stem cells and Cell Therapy; 2014

Human embryonic and induced pluripotent stem cells  (hESCs and hiPs, respectively) have great  capacity to differentiate in vitro and huge potenti  al to be used as cellular sources for regenerative  medicine. The basic fibroblast growth factor (bFGF)  is essential for survival, maintenance of  pluripotency and self-renewal of hESCs and hiPs. PI  3K and its most prominent effector, Akt,  regulate cell viability in many cell types. bFGF ac  tivates PI3K signaling pathway in these  pluripotent cells.  The aim of this work is to study the contribution o  f Akt in hESCs and hiPs viability.  hESCs lines WA01 (H1) - WA09 (H9) and hiPs line FN2  .1 (generated in our laboratory from  human foreskin fibroblasts) were cultured until con  fluence in the presence of bFGF. Then, cell  survival was analyzed in the presence of three stru  cturally unrelated Akt inhibitors. Inhibitors used  were: Akt inhibitor IV (specific of a kinase which  activates Akt), Akt inhibitor VIII (prevents bindin  g  of Akt to cell membrane) and GSK690693 (blocks ATP-  binding site of Akt). In particular, cell  viability was measured by the XTT colorimetric assa  y and Trypan blue exclusion test. Annexin V  exposure to outer cell membrane and DNA fragmentati  on are two of the most important criteria  used to indentify apoptotic cells, therefore change  s 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. Activ  ation of initiator and effector caspases (like  caspase-9 and caspase-3, respectively) is another h  allmark 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 abun  dance were also quantified by Western blot.  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 signific  antly 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 in H9, H1 and FN2.1 cells.  Moreover, Western blot analysis revealed the  activation of caspase-9, caspase-3 and PARP cleavag  e at different time points upon Akt inhibitors  treatment. Finally, no changes in Bcl-2 family memb  ers expression levels were detected by  Western Blot under the same experimental conditions  .  Taken together, we can conclude from the above results, that AKT activity is anti-apoptotic and  thus relevant to hESCs and hiPs survival.