Regulation of apoptosis related proteins in human pluripotent stem cells exposed to chemical hypoxia

ISAJA, LUCIANA; MUCCI, SOFÍA; VERA, JONATHAN S; RODRÍGUEZ VARELA, MARÍA SOLEDAD; MARAZITA, MARIELA; SEVLEVER, GUSTAVO EMILIO; SCASSA, MARÍA ELIDA; ROMORINI, LEONARDO

Mar del Plata

Congreso; LXIV Reunión Científica Anual de la Sociedad Argentina de Investigación Clínica SAIC; 2019

SAIC

Human embryonic and induced pluripotent stem cells (hESCs and hiPSCs) are self-renewing pluripotent cells (hPSCs) that can differentiate to a wide range of specialized cells. Notably, hPSCs tend to improve its undifferentiated state and its self-renewal under reduced oxygen conditions (hypoxia). Chemical compounds, like cobalt chloride (CoCl2), can be used in vitro to mimic hypoxic conditions by stabilizing the hypoxia- inducible factor 1a (HIF-1a). We have demonstrated that chemical hypoxia induces apoptosis of hPSCs. Herein, we aim to ascertain the molecular mechanisms underlying these events. First, we screened changes in the expression levels of 43 apoptosis-related proteins using a human apoptosis antibody array at 8 hours post-CoCl2 (250 μM) addition in hPSCs. Our results revealed that the extrinsic and the intrinsic pathway are involved in the apoptotic cascades activated by CoCl2 in hPSCs. Next, we quantified the expression levels of key Bcl-2 family members (not present in the apoptosis array) by Western blot and RT-qPCR in hPSCs after CoCl2 treatment (250 μM for 4, 8 and 24 h). We observed that Bnip-3, Mcl-1 and NOXA protein and mRNA expression levels were significantly upregulated at different time points upon hypoxia induction. In contrast, PUMA expression levels decreased. Importantly, p53 was also upregulated upon CoCl2 treatment (4 h). Additionally, we found that in hPSCs siRNA-mediated downregulation of HIF-1a did not revert the apoptosis rate induced by CoCl2 as judged by PI staining and Trypan blue dye exclusion data. However, the induction of Bnip-3 mRNA and protein expression levels was markedly impaired in HIF-1a-siRNA transfectants. Further supporting these findings, no changes in the percentage of apoptotic cells were observed when using Bnip3 siRNA. Taken together, our results indicate that the apoptosis induced by chemical hypoxia in hPSCs is HIF-1a and Bnip-3 independent and that both extrinsic and intrinsic apoptotic pathways could be involved.