EFFECT OF ACUTE HYPOXIA IN HUMAN PLURIPOTENT STEM CELLS SURVIVAL

JOHNATAN VERA; MARÍA ELIDA SCASSA; GUSTAVO SEVLEVER; SANTIAGO G. MIRIUKA; LEONARDO ROMORINI

Mar del Plata

Congreso; SAIC SAE SAFE 2016; 2016

SAIC

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. The first stages of embryogenesis evolve in a hypoxic environment. In this sense, it is well known that moderate hypoxia (5% O2) improves PSC self-renewal, pluripotency and cell survival. However, the effect of acute hypoxia (1% O2) in PSC has not been reported yet. The aim of this work was to explore the effect of acute hypoxia in PSC survival and cell death rate. To address this issue, hESCs (WA09/H9 line) and hiPSCs (FN2.1 line) were cultured under physical (1% O2) or chemical acute hypoxia conditions. The latter were generated with CoCl2 (250 nM) and dimethyl oxal glycine (DMOG) (100 nM and 1 mM) treatments, which stabilizes HIF-1α (key hypoxia inducible transcription factor) mimicking hypoxia. We observed an increase in the levels of HIF1-α target gen bnip-3 concomitant with a decrease (measured by XTT/PMS vital dye assay) in cell viability at 24 hours post-hypoxia induction. Importantly, at 24 hours post-hypoxia induction we observed the appearance of cell ballooning and detachment, hallmarks of programmed cell death, a reduction on the percentage of surviving cells (Trypan blue dye-exclusion assay), an increment on the percentage of apoptotic nuclei (Hoechst staining of nuclear DNA), and an increase in late apoptosis or necrosis (flow cytometry analysis with PI staining) and apoptotic DNA fragmentation (DNA oligomers quantified by ELISA) rates. Moreover, immunofluorescence analysis revealed activation of the effector Caspase-3 as soon as 6 hours after hypoxia induction. Collectively, in this study we demonstrated that acute physical (1% O2) and chemical hypoxia reduced human PSC survival and triggered apoptosis.