title: Noninvasive monitoring of oxidative stress in transplanted mesenchymal stromal cells.
abstract: OBJECTIVE
  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 p67(phox) subunit of nicotinamide adenine dinucleotide phosphate (NAD[P]H oxidase p67(phox)), an oxidative stress sensor was constructed, comprising the firefly luciferase (Fluc) reporter gene driven by the NAD(P)H p67(phox) promoter. MSCs cotransfected with NAD(P)H p67(phox)-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.
authors: Division of Cardiovascular Diseases, Department of Internal Medicine, Mayo Clinic, Rochester, MN 55905, USA.
authors: Peter J
authors: PJ
authors: Psaltis
authors: Karen M
authors: KM
authors: Peterson
authors: Rende
authors: R
authors: Xu
authors: Federico
authors: F
authors: Franchi
authors: Tyra
authors: T
authors: Witt
authors: Ian Y
authors: IY
authors: Chen
authors: Amir
authors: A
authors: Lerman
authors: Robert D
authors: RD
authors: Simari
authors: Sanjiv S
authors: SS
authors: Gambhir
authors: Martin
authors: M
authors: Rodriguez-Porcel
mesh_terms: D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D015374:Biosensing Techniques; D002470:Cell Survival; D058948:Cell Tracking / Q000379:methods; D002478:Cells, Cultured; D003497:Cyclic N-Oxides / Q000494:pharmacology; D003587:Cytomegalovirus / Q000235:genetics; D004195:Disease Models, Animal; D017930:Genes, Reporter; D049409:Luciferases, Firefly / Q000096:biosynthesis / Q000235:genetics; D049410:Luciferases, Renilla / Q000096:biosynthesis / Q000235:genetics; D008163:Luminescent Measurements; D045164:Mesenchymal Stem Cell Transplantation; D059630:Mesenchymal Stem Cells / Q000378:metabolism*; D057054:Molecular Imaging; D015428:Myocardial Reperfusion Injury / Q000235:genetics / Q000378:metabolism / Q000601:surgery*; D018384:Oxidative Stress / Q000187:drug effects; D010641:Phenotype; D010750:Phosphoproteins / Q000235:genetics; D011401:Promoter Regions, Genetic; D051381:Rats; D013113:Spin Labels; D013997:Time Factors; D014162:Transfection
chemical_list: D000975:Antioxidants; D003497:Cyclic N-Oxides; D010750:Phosphoproteins; D013113:Spin Labels; C079789:neutrophil cytosol factor 67K; D049410:Luciferases, Renilla; D049409:Luciferases, Firefly; C001803:tempol
references: Mayo Clin Proc. 2009 Oct;84(10):876-92
references: 19797777
references: Exp Cell Res. 2011 Jul 1;317(11):1541-7
references: 21376036
references: Cell Biol Int. 2009 Jun;33(6):665-74
references: 19376254
references: Stem Cells Dev. 2010 Sep;19(9):1437-48
references: 20109065
references: Stem Cells. 2010 Mar 31;28(3):555-63
references: 20073042
references: Gene Ther. 2010 Jul;17(7):827-38
references: 20237511
references: Nature. 2008 Feb 21;451(7181):937-42
references: 18288183
references: Circulation. 2003 Sep 16;108(11):1302-5
references: 12963637
references: Arterioscler Thromb Vasc Biol. 2012 Jan;32(1):92-102
references: 22034515
references: Stem Cell Rev Rep. 2013 Jun;9(3):281-302
references: 22529015
references: Circ Res. 2010 Feb 19;106(3):479-94
references: 20167944
references: Proc Natl Acad Sci U S A. 2001 Dec 4;98(25):14595-600
references: 11734653
references: Am J Physiol Heart Circ Physiol. 2010 May;298(5):H1320-9
references: 20154257
references: Circ Cardiovasc Imaging. 2008 Sep;1(2):94-103
references: 19808526
references: Radiology. 2009 Jul;252(1):117-27
references: 19366903
references: Circ Cardiovasc Imaging. 2010 Jan;3(1):77-85
references: 19920031
references: Circulation. 2008 Sep 30;118(14 Suppl):S121-9
references: 18824743
references: Stem Cells. 2009 Feb;27(2):375-82
references: 19038795
references: Nat Methods. 2007 Aug;4(8):641-3
references: 17618292
references: Life Sci. 2011 Jan 3;88(1-2):65-73
references: 21062632
references: Mol Imaging Biol. 2010 Jun;12(3):325-34
references: 20013064
references: Eur J Nucl Med Mol Imaging. 2012 Jan;39(1):165-81
references: 21901381
references: Circ Cardiovasc Imaging. 2012 Jan;5(1):94-101
references: 22135400
references: Stem Cells. 2009 Dec;27(12):3021-31
references: 19816949
references: PLoS One. 2011;6(9):e22949
references: 21912635
references: Stem Cell Res. 2011 May;6(3):215-25
references: 21421339
references: Circ Cardiovasc Imaging. 2008 Jul;1(1):6-13
references: 19808509