title: Genetic modification of embryonic stem cells with VEGF enhances cell survival and improves cardiac function.
abstract: Cardiac stem cell therapy remains hampered by acute donor cell death posttransplantation and the lack of reliable methods for tracking cell survival in vivo. We hypothesize that cells transfected with inducible vascular endothelial growth factor 165 (VEGF(165)) can improve their survival as monitored by novel molecular imaging techniques. Mouse embryonic stem (ES) cells were transfected with an inducible, bidirectional tetracycline (Bi-Tet) promoter driving VEGF(165) and renilla luciferase (Rluc). Addition of doxycycline induced Bi-Tet expression of VEGF(165) and Rluc significantly compared to baseline (p<0.05). Expression of VEGF(165) enhanced ES cell proliferation and inhibited apoptosis as determined by Annexin-V staining. For noninvasive imaging, ES cells were transduced with a double fusion (DF) reporter gene consisting of firefly luciferase and enhanced green fluorescence protein (Fluc-eGFP). There was a robust correlation between cell number and Fluc activity (R(2)=0.99). Analysis by immunostaining, histology, and RT-PCR confirmed that expression of Bi-Tet and DF systems did not affect ES cell self-renewal or pluripotency. ES cells were differentiated into beating embryoid bodies expressing cardiac markers such as troponin, Nkx2.5, and beta-MHC. Afterward, 5 x 10(5) cells obtained from these beating embryoid bodies or saline were injected into the myocardium of SV129 mice (n=36) following ligation of the left anterior descending (LAD) artery. Bioluminescence imaging (BLI) and echocardiography showed that VEGF(165) induction led to significant improvements in both transplanted cell survival and cardiac function (p<0.05). This is the first study to demonstrate imaging of embryonic stem cell-mediated gene therapy targeting cardiovascular disease. With further validation, this platform may have broad applications for current basic research and further clinical studies.
authors.affiliation: The Department of Radiology and Molecular Imaging Program at Stanford, Stanford University, Stanford, California, USA.
authors.forename: Xiaoyan
authors.initials: X
authors.lastname: Xie
authors.forename: Feng
authors.initials: F
authors.lastname: Cao
authors.forename: Ahmad Y
authors.initials: AY
authors.lastname: Sheikh
authors.forename: Zongjin
authors.initials: Z
authors.lastname: Li
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Connolly
authors.forename: Xuetao
authors.initials: X
authors.lastname: Pei
authors.forename: Ren-Ke
authors.initials: RK
authors.lastname: Li
authors.forename: Robert C
authors.initials: RC
authors.lastname: Robbins
authors.forename: Joseph C
authors.initials: JC
authors.lastname: Wu
mesh_terms: D000818:Animals; D017209:Apoptosis; D049109:Cell Proliferation; D002470:Cell Survival; D004318:Doxycycline / Q000494:pharmacology; D004452:Echocardiography / Q000379:methods; D053595:Embryonic Stem Cells / Q000166:cytology*; D005821:Genetic Techniques; D015316:Genetic Therapy / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006321:Heart / Q000502:physiology*; D051379:Mice; D009206:Myocardium / Q000473:pathology*; D011401:Promoter Regions, Genetic; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism*
chemical_list: D042461:Vascular Endothelial Growth Factor A; D049452:Green Fluorescent Proteins; D004318:Doxycycline