title: Optimizing reporter constructs for in vivo bioluminescence imaging of interferon-γ stimulated mesenchymal stromal cells.
abstract: Mesenchymal stromal cells (MSCs) are a promising treatment modality for a variety of diseases. Strategies to investigate the fate of MSCs in vivo are important to unravel their therapeutic mechanisms. However, currently available techniques are hampered by their low sensitivity. We therefore aimed to optimize in vivo bioluminescence imaging of MSCs. We compared MSCs transduced with firefly luciferase (Fluc) and transmembrane-bound Gaussia luciferase driven by the human cytomegalovirus, spleen focus-forming virus (SFFV), and elongation factor 1-α (EF1α) promoters. Although cytomegalovirus-transmembrane-bound Gaussia luciferase-transduced MSCs showed the highest light intensity in vitro, the signal was almost undetectable in vivo. Spleen focus-forming virus-Fluc-transduced MSCs revealed a bright signal in vivo, but transgene expression was silenced upon in vitro stimulation with interferon (IFN)-γ. Therefore, the SFFV promoter was replaced by the EF1α promoter. Light emission of Fluc under the control of EF1α was similar to SFFV-Fluc. Although EF1α-Fluc light emission was decreased tenfold in the presence of IFN-γ when compared with unstimulated MSCs, the bioluminescent signal could still be detected and was clearly distinguishable from untransduced MSCs. Furthermore, stimulation of MSCs with tumor necrosis factor-α hardly affected transgene expression in EF1α-Fluc-transduced MSCs. Thus, the use of the EF1α promoter partially overcomes silencing and allows in vivo bioluminescence imaging of IFN-γ-stimulated MSCs.
authors.affiliation: Department of Immunohematology and Bloodtransfusion, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: Jorge
authors.initials: J
authors.lastname: Perez-Galarza
authors.affiliation: Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, The Netherlands; Department of Nephrology, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: Françoise
authors.initials: F
authors.lastname: Carlotti
authors.affiliation: Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: Martijn J
authors.initials: MJ
authors.lastname: Rabelink
authors.affiliation: Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: Steve
authors.initials: S
authors.lastname: Cramer
authors.affiliation: Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: Rob C
authors.initials: RC
authors.lastname: Hoeben
authors.affiliation: Department of Immunohematology and Bloodtransfusion, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: Willem E
authors.initials: WE
authors.lastname: Fibbe
authors.affiliation: Department of Immunohematology and Bloodtransfusion, Leiden University Medical Center, Leiden, The Netherlands. Electronic address: m.van_pel@lumc.nl.
authors.forename: Melissa
authors.initials: M
authors.lastname: van Pel
mesh_terms: D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology*; D003587:Cytomegalovirus / Q000235:genetics / Q000378:metabolism; D017930:Genes, Reporter; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology*; D049409:Luciferases, Firefly / Q000096:biosynthesis* / Q000235:genetics; D008163:Luminescent Measurements / Q000379:methods*; D008297:Male; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D020648:Peptide Elongation Factor 1 / Q000235:genetics*; D011401:Promoter Regions, Genetic; D013155:Spleen Focus-Forming Viruses / Q000235:genetics / Q000378:metabolism; D014161:Transduction, Genetic
chemical_list: D000998:Antiviral Agents; D020648:Peptide Elongation Factor 1; D007371:Interferon-gamma; D049409:Luciferases, Firefly