Noninvasive Monitoring of Oxidative Stress in Transplanted Mesenchymal Stromal Cells

Abstract

Objectives We aimed 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 upregulation of the pro-oxidant enzyme 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 co-transfected with NAD(P)H p67phox-Fluc and a cell viability reporter gene (CMV-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 bioluminescence imaging. Compared to 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 was corroborated by ex vivo luminometry after correcting for renilla luciferase (Rluc) activity as a measure of viable MSC number (Fluc:Rluc ratio 0.011 ± 0.003 for sham versus 0.026 ± 0.004 for IR at 48 h, P<0.05). Furthermore, in IR animals that received MSCs preconditioned with an anti-oxidant 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.

Translational applications of molecular imaging in cardiovascular disease and stem cell therapy

“…Moreover, staining histological sections with annexin V also got a consistent result [28]. Meanwhile, other targets, including 64 Cu-TNP [29], MPO(Gd) [30], 123 I-BMIPP [31], 111 I-VEGF 121 [32], EP-2104R [33], have been used to assess angiogenesis processes in atherosclerosis by established radiotracers.…”

“…The conditions of some molecules can reflect the state of disease progression after stem cells delivery. For instance, reporter gene imaging allows assessment of changes in the oxidative status of mesenchymal stem cells (MSCs) after delivery to ischemic myocardium in a rat model of myocardial ischemia/reperfusion [64]. So imaging of these molecules, such as imaging of integrin a v b 3, exposed phosphatidylserine, glucose metabolism and extracellular matrix remodeling, provides a better understanding of molecular changes after stem cells therapy (Fig.…”

Enhancement of cell adhesion, retention, and survival of HUVEC/cbMSC aggregates that are transplanted in ischemic tissues by concurrent delivery of an antioxidant for therapeutic angiogenesis

“…To evaluate the oxidative stress in tissues, test animals were euthanized on the first day following treatment, and their quadriceps muscles were harvested and processed for immunostaining. Tissue sections were separately incubated with primary antibodies against oxidative-damage markers 8-hydroxy-2'-deoxyguanosine (8-OHdG; JaICA, Shizuoka, Japan) and 4-hydroxynonenal (4-HNE) [31,32], visualized by fluorophore-conjugated secondary antibodies (Life Technologies), counterstained with DAPI, and then observed using a CLSM.…”

Noninvasive Monitoring of the Mitochondrial Function in Mesenchymal Stromal Cells

“…In fact, bioluminescence imaging (BLI) has been used to study the kinetics of the viability of myocardial-implanted cells in living animals [27]. Furthermore, we recently reported that reporter gene imaging can also be used to monitor specific biological processes using conditional transgenes whose expression is regulated by pathway-specific promoters [32]. …”