Chemical hypoxia induces apoptosis of human pluripotent stem cells by a NOXA-mediated HIF-1\u03b1 and HIF-2\u03b1 independent mechanism

Luciana Isaja,Olivia Morris-Hanon,Leonardo Romorini,Mariela Marazita,Jonathan Vera,Sofía Mucci,María Soledad Rodríguez-Varela,Guillermo Agustín Videla-Richardson,Gustavo Emilio Sevlever,María Elida Scassa

doi:10.1038/s41598-020-77792-7

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.

Highlights

Human embryonic stem cells and human induced pluripotent stem cells are self-renewing pluripotent stem cells that can differentiate and give rise to all cell types derived from the three germ layers, endoderm, mesoderm and ectoderm
Hypoxia was induced in hESCs line WA09 (H9) Human embryonic stem cells (hESCs) and FN2.1 human induced pluripotent stem cells (hiPSCs) grown on Vitronectin coated cell culture dishes with fully defined Essential E8 medium (E8) by a chemical method
Key regulators of the hypoxia response are Hypoxia-inducible transcription factor 1α (HIF-1α) and HIF-2α. These transcription factors exert a complex role regulating more than 100 genes including glycolytic enzymes and survival factors, which are required to cope with low oxygen tensions

Summary

Introduction

Human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) are self-renewing pluripotent stem cells (hPSCs) that can differentiate and give rise to all cell types derived from the three germ layers, endoderm, mesoderm and ectoderm. HPSCs in vitro culture is still usually performed under ambient air (O2 concentration of approximately 21%) enriched with 5% CO2, given that modular hypoxia incubator chambers are not frequently available in many cell culture facilities To overcome this limitation, chemical compounds that inhibit prolyl hydroxylase domain-containing enzymes, like cobalt chloride (CoCl2) and dimethyloxalylglycine (DMOG) can be used in vitro to mimic hypoxic conditions by stabilizing HIF-1α and generating R OS12–14. Chemical compounds that inhibit prolyl hydroxylase domain-containing enzymes, like cobalt chloride (CoCl2) and dimethyloxalylglycine (DMOG) can be used in vitro to mimic hypoxic conditions by stabilizing HIF-1α and generating R OS12–14 In this regard, until the present, the effects of chemical induced hypoxia on hPSCs viability have not been studied. We demonstrated that chemical hypoxia triggered apoptosis in hPSCs via a NOXA-mediated HIF-1α and HIF-2α independent mechanism

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