Chemical hypoxia induces apoptosis of human pluripotent stem cells by a NOXA-mediated HIF-1α and HIF-2α independent mechanism

Abstract

Human embryonic and induced pluripotent stem cells (hESCs and hiPSCs) are self-renewing human pluripotent stem cells (hPSCs) that can differentiate to a wide range of specialized cells. Notably, hPSCs enhance their undifferentiated state and self-renewal properties in hypoxia (5% O2). Although thoroughly analyzed, hypoxia implication in hPSCs death is not fully determined. In order to evaluate the effect of chemically mimicked hypoxia on hPSCs cell survival, we analyzed changes in cell viability and several aspects of apoptosis triggered by CoCl2 and dimethyloxalylglycine (DMOG). Mitochondrial function assays revealed a decrease in cell viability at 24 h post-treatments. Moreover, we detected chromatin condensation, DNA fragmentation and CASPASE-9 and 3 cleavages. In this context, we observed that P53, BNIP-3, and NOXA protein expression levels were significantly up-regulated at different time points upon chemical hypoxia induction. However, only siRNA-mediated downregulation of NOXA but not HIF-1α, HIF-2α, BNIP-3, and P53 did significantly affect the extent of cell death triggered by CoCl2 and DMOG in hPSCs. In conclusion, chemically mimicked hypoxia induces hPSCs cell death by a NOXA-mediated HIF-1α and HIF-2α independent mechanism.

Acute severe hypoxia induces apoptosis of human pluripotent stem cells by a HIF-1α and P53 independent mechanism

“…hPSCs are very susceptible to undergo apoptosis due to their higher state of mitochondrial priming, a lowered cell intrinsic threshold for initiating mitochondrial-induced apoptosis, based on the balance between pro- and anti-apoptotic protein members of the BCL-2 family 5 , 14 , 20 . Bearing this in mind, we performed western blot assays to analyze the expression levels of crucial BCL-2 family members in hPSCs at 4-, 8- and 24-h post-hypoxia (1% O 2 ).…”

“…Thus, elucidating the mechanisms involved in hypoxia-induced apoptosis may help to control hPSC fate, and minimize cell death within hypoxic niches. To face this obstacle, we recently demonstrated that CoCl 2 , a widely used chemical surrogate of hypoxia, triggered apoptotic cell death in hPSCs via a NOXA-mediated HIF-1α and HIF-2α independent mechanism 5 . Our findings suggest that MCL-1 could regulate hPSC survival after exposure to 1% O 2 and that depending on the hypoxia inducer, alternative gene expression programs become operative in hPSCs.…”

“…Total RNA was extracted from hPSCs with TRIzol (Thermo Scientific) and cDNA was synthesized from 500 ng of total RNA with 15 mM of random hexamers and MMLV reverse transcriptase (Promega), according to manufacturer's instructions as previously described 5 , 56 . For qPCR studies, cDNA samples were diluted fivefold and PCR amplification and analysis were performed with StepOnePlus Real-Time PCR System (PE Applied Biosystems).…”

“…In normoxia, HIF-1α and HIF-2α are polyubiquitinated and targeted for proteasomal degradation. Instead, in low oxygen concentrations, they are stabilized 5 . Once stabilized, dimerize with HIF-1β, which is constitutively expressed, and regulate the transcription of more than 100 genes (e. g. glycolytic enzymes and survival factors) required to cope with low oxygen tensions 6 , 7 .…”

“…Acute severe hypoxia may induce hPSC apoptosis as these cells are mitochondrial primed and thought highly sensitive to stressful stimuli 13 , 14 . In this sense, we and others have reported that chemical compounds routinely used in vitro to mimic hypoxia by HIF-1α stabilization and ROS generation, like cobalt chloride (CoCl 2 ) and dimethyloxalylglycine (DMOG) 15 – 17 , induce apoptosis and necrosis of hPSCs and mouse embryonic stem cells (mESCs) 5 , 15 .…”