Efficient Recombinase-Mediated Cassette Exchange in hPSCs to Study the Hepatocyte Lineage Reveals AAVS1 Locus-Mediated Transgene Inhibition
Authors: Catherine M Verfaillie
Kim Vanuytsel
Rangarajan Sambathkumar
Kristel Eggermont
Sylwia Bobis-Wozowicz
Ruben Boon
Qing Cai
Jolien Vanhove
Wenting Guo
Pieter Berckmans
Veerle Vanslembrouck
Tom Struys
Laura Ordov√°s
Ludo Van Den Bosch
Esther Wolfs
Susanna Raitano
Yemiao Chen
Mariaelena Pistoni
Toni Cathomen
Yaakov Nahmias
Nicky Helsen
B√©la Z Schmidt
Journal: Stem cell reports (2015)
Discipline: MOLECULAR BIOLOGY & GENETICS
Keywords: transgenesis; safe harbor loci; isogenic context; hPSCs; FLPe recombinase
Affiliations: 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: ., Stem Cell Institute, KU Leuven, Leuven 3000, Belgium; Department of Development and Regeneration, Stem Cell Biology and Embryology, KU Leuven, Leuven 3000, Belgium., Group of Morphology, Biomedical Research Institute, Hasselt University, Diepenbeek 3590, Belgium., 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., Institute for Cell and Gene Therapy, University Medical Center Freiburg, Freiburg 79108, Germany; Center for Chronic Immunodeficiency, University Medical Center Freiburg, Freiburg 79108, Germany., Switch Laboratory, VIB, Leuven 3000, Belgium; Department of Cellular and Molecular Medicine, Switch Laboratory, KU Leuven, Leuven 300, Belgium., Department of Cell and Developmental Biology, Hebrew University of Jerusalem, Jerusalem 91904, Israel; Grass Center for Bioengineering, Hebrew University of Jerusalem, Jerusalem 91904, Israel.

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. 

Retracted: No

PubPeer comments:
 This article has a correction, published on November 20, 2015. It reads:  The karyotype pictures of iPSC clones iMCL#3 and iMCL#11 shown in Figure S1D in the original Supplemental Material were incorrect and have been replaced online. This graphical error does not affect the results and conclusions of the article. The authors apologize for this error. ,