Live reporting for hypoxia: Hypoxia sensor–modified mesenchymal stem cells as in vitro reporters

Abstract

Natural oxygen gradients occur in tissues of biological organisms and also in the context of three-dimensional (3D) in vitro cultivation. Oxygen diffusion limitation and metabolic oxygen consumption by embedded cells produce areas of hypoxia in the tissue/matrix. However, reliable systems to detect oxygen gradients and cellular response to hypoxia in 3D cell culture systems are still missing. In this study, we developed a system for visualization of oxygen gradients in 3D using human adipose tissue-derived mesenchymal stem cells (hAD-MSCs) modified to stably express a fluorescent genetically engineered hypoxia sensor HRE-dUnaG. Modified cells retained their stem cell characteristics in terms of proliferation and differentiation capacity. The hypoxia-reporter cells were evaluated by fluorescence microscopy and flow cytometry under variable oxygen levels (2.5%, 5%, and 7.5% O 2). We demonstrated that reporter hAD-MSCs output is sensitive to different oxygen levels and displays fast decay kinetics after reoxygenation. Additionally, the reporter cells were encapsulated in bulk hydrogels with a variable cell number, to investigate the sensor response in model 3D cell culture applications. The use of hypoxia-reporting cells based on MSCs represents a valuable tool for approaching the genuine in vivo cellular microenvironment and will allow a better understanding of the regenerative potential of AD-MSCs.

Hypoxia Onset in Mesenchymal Stem Cell Spheroids: Monitoring With Hypoxia Reporter Cells

“…As a result, only mean fluorescence of the entire spheroid population can be obtained. As shown in our earlier works, the fluorescent signal of the hypoxia reporter hAD-MSCs at 2.5% O 2 in 2D cultures reached an average signal of ∼1.3 × 10 6 RFU (Schmitz et al, 2020). The detected average signal in this work is twice weaker (max.…”

“…hAD-MSCs were used for lentiviral transduction, to stably integrate HRE-dUnaG (Erapaneedi et al, 2016) sequence. Efficient transduction with HRE-dUnaG and oxygen-dependend UnaG expression was shown in our previous work (Schmitz et al, 2020). Surface antigen expression of hypoxia reporter modified MSCs was evaluated by flow cytometry.…”

“…Hypoxia reporter MSCs were created as described earlier (Schmitz et al, 2020). Briefly, hAD-MSC were isolated from adipose tissue after abdominoplasty applying the protocol of Zhu et al (2008).…”

“…The system relies on the expression of fluorescent UnaG protein (Kumagai et al, 2013), whose maturation process is independent from presence of molecular oxygen. While this construct provides a great tool for hypoxia research in general, the application of hypoxia reporter cells is invaluable for 3D cell culture applications (Schmitz et al, 2020). Finally, a non-invasive hypoxia monitoring allows the live analysis of the hypoxic state of single cells within complex constructs.…”

Biosensors to Monitor Cell Activity in 3D Hydrogel-Based Tissue Models

“…One of the most important chemical cues to maintain cellular phenotype and function in vitro is surely the oxygen concentration, which is essential in the energy metabolism of the cells. O 2 is a potent modulator of cell function and a transcriptional regulator for over 300 genes responsible for tissue homeostasis and maintenance [131,132]. Moreover, among the great variety of biochemical signals, O 2 plays a pivotal role in modulating mammalian cell mechanisms both in healthy and diseased states [133].…”