Original Research
Noninvasive Monitoring of Oxidative Stress in Transplanted Mesenchymal Stromal Cells

https://doi.org/10.1016/j.jcmg.2012.11.018Get rights and content
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Objectives

The goal of this study was to validate a pathway-specific reporter gene that could be used to noninvasively image the oxidative status of progenitor cells.

Background

In cell therapy studies, reporter gene imaging plays a valuable role in the assessment of cell fate in living subjects. After myocardial injury, noxious stimuli in the host tissue confer oxidative stress to transplanted cells that may influence their survival and reparative function.

Methods

Rat mesenchymal stromal cells (MSCs) were studied for phenotypic evidence of increased oxidative stress under in vitro stress. On the basis of their up-regulation of the pro-oxidant enzyme p67phox subunit of nicotinamide adenine dinucleotide phosphate (NAD[P]H oxidase p67phox), an oxidative stress sensor was constructed, comprising the firefly luciferase (Fluc) reporter gene driven by the NAD(P)H p67phox promoter. MSCs cotransfected with NAD(P)H p67phox–Fluc and a cell viability reporter gene (cytomegalovirus–Renilla luciferase) were studied under in vitro and in vivo pro-oxidant conditions.

Results

After in vitro validation of the sensor during low-serum culture, transfected MSCs were transplanted into a rat model of myocardial ischemia/reperfusion (IR) and monitored by using bioluminescence imaging. Compared with sham controls (no IR), cardiac Fluc intensity was significantly higher in IR rats (3.5-fold at 6 h, 2.6-fold at 24 h, 5.4-fold at 48 h; p < 0.01), indicating increased cellular oxidative stress. This finding was corroborated by ex vivo luminometry after correcting for Renilla luciferase activity as a measure of viable MSC number (Fluc:Renilla luciferase ratio 0.011 ± 0.003 for sham vs. 0.026 ± 0.004 for IR at 48 h; p < 0.05). Furthermore, in IR animals that received MSCs preconditioned with an antioxidant agent (tempol), Fluc signal was strongly attenuated, substantiating the specificity of the oxidative stress sensor.

Conclusions

Pathway-specific reporter gene imaging allows assessment of changes in the oxidative status of MSCs after delivery to ischemic myocardium, providing a template to monitor key biological interactions between transplanted cells and their host environment in living subjects.

Key Words

bioluminescence
mesenchymal stem cells
NAD(P)H oxidase
oxidative stress
reporter gene

Abbreviations and Acronyms

BLI
bioluminescence imaging
CMV
cytomegalovirus
FBS
fetal bovine serum
Fluc
firefly luciferase
IR
ischemia/reperfusion
LV
left ventricular
MSC
mesenchymal stromal cell
NAD(P)H
nicotinamide adenine dinucleotide phosphate
PET
positron emission tomography
ROS
reactive oxygen species
TSTA
2-step transcriptional amplification

Cited by (0)

The luminometer used was obtained through a grant from Turner BioSystems. This work was supported in part by the National Institutes of Health (HL 88048, Dr. Rodriguez-Porcel) and the Mayo Foundation (Dr. Rodriguez-Porcel). Dr. Psaltis has received funding from the National Health and Medical Research Council of Australia. All other authors have reported that they have no relationships relevant to the contents of this paper to disclose.