Exploring the impact of hypoxia mimetic agents on multipotent stem cell biology

Abstract

Oxygen is an important molecule in life and is essential for a broad spectrum of physiological reactions that include, but are not restricted to, cell metabolism, respiration and growth. Under physiological conditions, oxygen levels vary from one tissue to another ranging from 0.002% to 10% and substantially lower than the atmospheric level of 21 % O2. Hypoxia is defined as a state of reduced O2 supplied to cells or tissues when compared to their normal physiological levels. Hypoxia mimetic agents (HMAs) are chemical used to induce pharmacological hypoxia without affecting environmental oxygen levels per se. The name HMA is routinely applied as these agents will activate the family of transcription factors which respond to reduced oxygen availability, Hypoxia Inducible Factors (HIF), which is taken as a surrogate indicator for hypoxia. These agents have been proposed as a cheaper alternative to engineered oxygen control measures including tri-gas incubators and workstation approaches. Multipotent stem cells (e.g. neuronal PC12 and human mesenchymal stem cells (hMSCs)) due their ability to differentiate into various cell types provide a means to develop better understanding of tissue development, repair, and protection. In addition, they provide better therapeutic perspectives and opportunities for the treatment of many diseases. Physiological oxygen plays a key role in the maintenance of cellular proliferation, behaviour and biology both in vivo and when mimiced in vitro. Physiological oxygen and hypoxia are routinely confused creating additional complexity in defining the role of oxygen in cell behaviours. This work has therefore evaluated the role of a panel of well-established HMAs (CoCl2, DFO, DMOG) and new agent (IOX2) vs. different reduced oxygen culture conditions. PC12 and hMSCs were both used to examinine the roles of HMAs on proliferation, metabolic activity, HIFs expression, nitroreductase activity, oxidative stress, apoptosis, death/necrosis and mitochondrial dynamic (burden, action potential and genome copy number) in comparison to physiological normoxia and intermittent hypoxia. HMAs induced HIF expression, apoptosis, trapped cell at G0/G1 phase and induced both ROS formation and nitroreductase activity in a manner that was not consistent with a reduced oxygen culture condition alone. Mitochondrial burden, mitochondrial action potential, and mitochondrial genome number also changed in response to HMA exposure in a manner that was indenependent of the oxygen culture conditions tested. In summary, HMAs do not provide an accurate replication of engineered oxygen control measures in PC12 and hMSC biology. This is reflected in biological alterations impacting on cell yield, behaviour and biology.