title: Bioluminescent imaging of genetically selected induced pluripotent stem cell-derived cardiomyocytes after transplantation into infarcted heart of syngeneic recipients.
abstract: Cell loss after transplantation is a major limitation for cell replacement approaches in regenerative medicine. To assess the survival kinetics of induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CM) we generated transgenic murine iPSC lines which, in addition to CM-specific expression of puromycin N-acetyl-transferase and enhanced green fluorescent protein (EGFP), also constitutively express firefly luciferase (FLuc) for bioluminescence (BL) in vivo imaging. While undifferentiated iPSC lines generated by random integration of the transgene into the genome retained stable FLuc activity over many passages, the BL signal intensity was strongly decreased in purified iPS-CM compared to undifferentiated iPSC. Targeted integration of FLuc-expression cassette into the ROSA26 genomic locus using zinc finger nuclease (ZFN) technology strongly reduced transgene silencing in iPS-CM, leading to a several-fold higher BL compared to iPS-CM expressing FLuc from random genomic loci. To investigate the survival kinetics of iPS-CM in vivo, purified CM obtained from iPSC lines expressing FLuc from a random or the ROSA26 locus were transplanted into cryoinfarcted hearts of syngeneic mice. Engraftment of viable cells was monitored by BL imaging over 4 weeks. Transplanted iPS-CM were poorly retained in the myocardium independently of the cell line used. However, up to 8% of cells survived for 28 days at the site of injection, which was confirmed by immunohistological detection of EGFP-positive iPS-CM in the host tissue. Transplantation of iPS-CM did not affect the scar formation or capillary density in the periinfarct region of host myocardium. This report is the first to determine the survival kinetics of drug-selected iPS-CM in the infarcted heart using BL imaging and demonstrates that transgene silencing in the course of iPSC differentiation can be greatly reduced by employing genome editing technology. FLuc-expressing iPS-CM generated in this study will enable further studies to reduce their loss, increase long-term survival and functional integration upon transplantation.
authors.affiliation: Institute for Neurophysiology, Medical Faculty, University of Cologne, Cologne, Germany.
authors.forename: Vera
authors.initials: V
authors.lastname: Lepperhof
authors.affiliation: Institute for Neurophysiology, Medical Faculty, University of Cologne, Cologne, Germany.
authors.forename: Olga
authors.initials: O
authors.lastname: Polchynski
authors.affiliation: In-vivo-NMR Laboratory, Max Planck Institute for Neurological Research, Cologne, Germany.
authors.forename: Klaus
authors.initials: K
authors.lastname: Kruttwig
authors.affiliation: In-vivo-NMR Laboratory, Max Planck Institute for Neurological Research, Cologne, Germany.
authors.forename: Chantal
authors.initials: C
authors.lastname: Brüggemann
authors.affiliation: Department of Cardiothoracic Surgery, Heart Center of the University of Cologne, Cologne, Germany; Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany.
authors.forename: Klaus
authors.initials: K
authors.lastname: Neef
authors.affiliation: Department of Cardiothoracic Surgery, Heart Center of the University of Cologne, Cologne, Germany.
authors.forename: Florian
authors.initials: F
authors.lastname: Drey
authors.affiliation: Institute for Neurophysiology, Medical Faculty, University of Cologne, Cologne, Germany.
authors.forename: Yunjie
authors.initials: Y
authors.lastname: Zheng
authors.affiliation: Max Planck Institute for Metabolism Research and Institute for Genetics, Cologne, Germany.
authors.forename: Justus P
authors.initials: JP
authors.lastname: Ackermann
authors.affiliation: Department of Cardiothoracic Surgery, Heart Center of the University of Cologne, Cologne, Germany; Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany.
authors.forename: Yeong-Hoon
authors.initials: YH
authors.lastname: Choi
authors.affiliation: Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany; Max Planck Institute for Metabolism Research and Institute for Genetics, Cologne, Germany; Cologne Cluster of Excellence in Cellular Stress Responses in Aging-Associated Diseases (CECAD), Cologne, Germany.
authors.forename: Thomas F
authors.initials: TF
authors.lastname: Wunderlich
authors.affiliation: In-vivo-NMR Laboratory, Max Planck Institute for Neurological Research, Cologne, Germany.
authors.forename: Mathias
authors.initials: M
authors.lastname: Hoehn
authors.affiliation: Institute for Neurophysiology, Medical Faculty, University of Cologne, Cologne, Germany.
authors.forename: Jürgen
authors.initials: J
authors.lastname: Hescheler
authors.affiliation: Institute for Neurophysiology, Medical Faculty, University of Cologne, Cologne, Germany.
authors.forename: Tomo
authors.initials: T
authors.lastname: Sarić
mesh_terms: D000818:Animals; D002454:Cell Differentiation; D002460:Cell Line; D015870:Gene Expression; D020868:Gene Silencing; D017930:Genes, Reporter; D056426:Genetic Loci; D057026:Induced Pluripotent Stem Cells / Q000166:cytology* / Q000378:metabolism; D008163:Luminescent Measurements / Q000379:methods*; D008297:Male; D051379:Mice; D057054:Molecular Imaging; D009203:Myocardial Infarction / Q000175:diagnosis* / Q000401:mortality / Q000473:pathology / Q000628:therapy*; D032383:Myocytes, Cardiac / Q000166:cytology* / Q000378:metabolism / Q000637:transplantation*; D011401:Promoter Regions, Genetic; D022661:RNA, Untranslated / Q000235:genetics; D014161:Transduction, Genetic; D019076:Transgenes
chemical_list: C110853:Gt(ROSA)26Sor non-coding RNA, mouse; D022661:RNA, Untranslated