title: Efficient Recombinase-Mediated Cassette Exchange in hPSCs to Study the Hepatocyte Lineage Reveals AAVS1 Locus-Mediated Transgene Inhibition.
abstract: Tools for rapid and efficient transgenesis in "safe harbor" loci in an isogenic context remain important to exploit the possibilities of human pluripotent stem cells (hPSCs). We created hPSC master cell lines suitable for FLPe recombinase-mediated cassette exchange (RMCE) in the AAVS1 locus that allow generation of transgenic lines within 15 days with 100% efficiency and without random integrations. Using RMCE, we successfully incorporated several transgenes useful for lineage identification, cell toxicity studies, and gene overexpression to study the hepatocyte lineage. However, we observed unexpected and variable transgene expression inhibition in vitro, due to DNA methylation and other unknown mechanisms, both in undifferentiated hESC and differentiating hepatocytes. Therefore, the AAVS1 locus cannot be considered a universally safe harbor locus for reliable transgene expression in vitro, and using it for transgenesis in hPSC will require careful assessment of the function of individual transgenes.
authors.affiliation: Stem Cell Institute, KU Leuven, Leuven 3000, Belgium; Department of Development and Regeneration, Stem Cell Biology and Embryology, KU Leuven, Leuven 3000, Belgium. Electronic address: laura.ordovas@med.kuleuven.be.
authors.forename: Laura
authors.initials: L
authors.lastname: Ordovás
authors.affiliation: Stem Cell Institute, KU Leuven, Leuven 3000, Belgium; Department of Development and Regeneration, Stem Cell Biology and Embryology, KU Leuven, Leuven 3000, Belgium.
authors.forename: Ruben
authors.initials: R
authors.lastname: Boon
authors.affiliation: Stem Cell Institute, KU Leuven, Leuven 3000, Belgium; Department of Development and Regeneration, Stem Cell Biology and Embryology, KU Leuven, Leuven 3000, Belgium.
authors.forename: Mariaelena
authors.initials: M
authors.lastname: Pistoni
authors.affiliation: Stem Cell Institute, KU Leuven, Leuven 3000, Belgium; Department of Development and Regeneration, Stem Cell Biology and Embryology, KU Leuven, Leuven 3000, Belgium.
authors.forename: Yemiao
authors.initials: Y
authors.lastname: Chen
authors.affiliation: Group of Morphology, Biomedical Research Institute, Hasselt University, Diepenbeek 3590, Belgium.
authors.forename: Esther
authors.initials: E
authors.lastname: Wolfs
authors.affiliation: Leuven Research Institute for Neuroscience and Disease (LIND), Leuven 3000, Belgium; Department of Neurosciences, Experimental Neurology, KU Leuven, Leuven 3000, Belgium; Laboratory for Neurobiology, VIB-Vesalius Research Center, Leuven 3000, Belgium.
authors.forename: Wenting
authors.initials: W
authors.lastname: Guo
authors.affiliation: Stem Cell Institute, KU Leuven, Leuven 3000, Belgium; Department of Development and Regeneration, Stem Cell Biology and Embryology, KU Leuven, Leuven 3000, Belgium.
authors.forename: Rangarajan
authors.initials: R
authors.lastname: Sambathkumar
authors.affiliation: Institute for Cell and Gene Therapy, University Medical Center Freiburg, Freiburg 79108, Germany; Center for Chronic Immunodeficiency, University Medical Center Freiburg, Freiburg 79108, Germany.
authors.forename: Sylwia
authors.initials: S
authors.lastname: Bobis-Wozowicz
authors.affiliation: Stem Cell Institute, KU Leuven, Leuven 3000, Belgium; Department of Development and Regeneration, Stem Cell Biology and Embryology, KU Leuven, Leuven 3000, Belgium.
authors.forename: Nicky
authors.initials: N
authors.lastname: Helsen
authors.affiliation: Stem Cell Institute, KU Leuven, Leuven 3000, Belgium; Department of Development and Regeneration, Stem Cell Biology and Embryology, KU Leuven, Leuven 3000, Belgium.
authors.forename: Jolien
authors.initials: J
authors.lastname: Vanhove
authors.affiliation: Stem Cell Institute, KU Leuven, Leuven 3000, Belgium; Department of Development and Regeneration, Stem Cell Biology and Embryology, KU Leuven, Leuven 3000, Belgium.
authors.forename: Pieter
authors.initials: P
authors.lastname: Berckmans
authors.affiliation: Stem Cell Institute, KU Leuven, Leuven 3000, Belgium; Department of Development and Regeneration, Stem Cell Biology and Embryology, KU Leuven, Leuven 3000, Belgium.
authors.forename: Qing
authors.initials: Q
authors.lastname: Cai
authors.affiliation: Stem Cell Institute, KU Leuven, Leuven 3000, Belgium; Department of Development and Regeneration, Stem Cell Biology and Embryology, KU Leuven, Leuven 3000, Belgium.
authors.forename: Kim
authors.initials: K
authors.lastname: Vanuytsel
authors.affiliation: Stem Cell Institute, KU Leuven, Leuven 3000, Belgium; Department of Development and Regeneration, Stem Cell Biology and Embryology, KU Leuven, Leuven 3000, Belgium.
authors.forename: Kristel
authors.initials: K
authors.lastname: Eggermont
authors.affiliation: Stem Cell Institute, KU Leuven, Leuven 3000, Belgium; Department of Development and Regeneration, Stem Cell Biology and Embryology, KU Leuven, Leuven 3000, Belgium.
authors.forename: Veerle
authors.initials: V
authors.lastname: Vanslembrouck
authors.affiliation: Switch Laboratory, VIB, Leuven 3000, Belgium; Department of Cellular and Molecular Medicine, Switch Laboratory, KU Leuven, Leuven 300, Belgium.
authors.forename: Béla Z
authors.initials: BZ
authors.lastname: Schmidt
authors.affiliation: Stem Cell Institute, KU Leuven, Leuven 3000, Belgium; Department of Development and Regeneration, Stem Cell Biology and Embryology, KU Leuven, Leuven 3000, Belgium.
authors.forename: Susanna
authors.initials: S
authors.lastname: Raitano
authors.affiliation: Leuven Research Institute for Neuroscience and Disease (LIND), Leuven 3000, Belgium; Department of Neurosciences, Experimental Neurology, KU Leuven, Leuven 3000, Belgium; Laboratory for Neurobiology, VIB-Vesalius Research Center, Leuven 3000, Belgium.
authors.forename: Ludo
authors.initials: L
authors.lastname: Van Den Bosch
authors.affiliation: Department of Cell and Developmental Biology, Hebrew University of Jerusalem, Jerusalem 91904, Israel; Grass Center for Bioengineering, Hebrew University of Jerusalem, Jerusalem 91904, Israel.
authors.forename: Yaakov
authors.initials: Y
authors.lastname: Nahmias
authors.affiliation: Institute for Cell and Gene Therapy, University Medical Center Freiburg, Freiburg 79108, Germany; Center for Chronic Immunodeficiency, University Medical Center Freiburg, Freiburg 79108, Germany.
authors.forename: Toni
authors.initials: T
authors.lastname: Cathomen
authors.affiliation: Group of Morphology, Biomedical Research Institute, Hasselt University, Diepenbeek 3590, Belgium.
authors.forename: Tom
authors.initials: T
authors.lastname: Struys
authors.affiliation: Stem Cell Institute, KU Leuven, Leuven 3000, Belgium; Department of Development and Regeneration, Stem Cell Biology and Embryology, KU Leuven, Leuven 3000, Belgium. Electronic address: catherine.verfaillie@med.kuleuven.be.
authors.forename: Catherine M
authors.initials: CM
authors.lastname: Verfaillie
mesh_terms: D002478:Cells, Cultured; D019175:DNA Methylation; D000229:Dependovirus / Q000235:genetics; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism*; D020868:Gene Silencing; D018390:Gene Targeting / Q000379:methods*; D056426:Genetic Loci; D022781:Hepatocytes / Q000166:cytology* / Q000378:metabolism; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism*; D045522:Recombinases / Q000235:genetics / Q000378:metabolism*; D019076:Transgenes
chemical_list: D045522:Recombinases