title: Modified lentiviral LTRs allow Flp recombinase-mediated cassette exchange and in vivo tracing of "factor-free" induced pluripotent stem cells.
abstract: Methods for generating induced pluripotent stem cells (iPSCs) for disease modeling and cell therapies have progressed from integrating vectors to transient delivery of reprogramming factors, avoiding permanent genomic modification. A major limitation of unmodified iPSCs is the assessment of their distribution and contribution to adverse reactions in autologous cell therapy. Here, we report that polycistronic lentiviral vectors with single Flp recombinase (Flp) recognition target (FRT) sites can be used to generate murine iPSCs that are devoid of the reprogramming cassette but carry an intergenic 300-bp long terminal repeat sequence. Performing quantitative polymerase chain reaction on this marker, we could determine genetic identity and tissue contribution of iPSC-derived teratomas in mice. Moreover, we generated iPSCs carrying heterospecific FRT twin sites, enabling excision and recombinase-mediated cassette exchange (RMCE) of the reprogramming cassette for another expression unit of choice. Following screening of iPSCs for "safe harbor" integration sites, expression cassettes were introduced by RMCE into various previously silenced loci of selected single-copy iPSCs. Analysis of DNA methylation showed that RMCE reverted the local epigenetic signature, which allowed transgene expression in undifferentiated iPSCs and in differentiated progeny. These findings support the concept of creating clonotypically defined exchangeable and traceable pluripotent stem cells for disease research and cell therapy.
authors.affiliation: Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany.
authors.forename: Johannes
authors.initials: J
authors.lastname: Kuehle
authors.affiliation: Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany.
authors.forename: Soeren
authors.initials: S
authors.lastname: Turan
authors.affiliation: Research Group Translational Hepatology and Stem Cell Biology, REBIRTH Cluster of Excellence, Hannover Medical School, Hannover, Germany.
authors.forename: Tobias
authors.initials: T
authors.lastname: Cantz
authors.affiliation: Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany.
authors.forename: Dirk
authors.initials: D
authors.lastname: Hoffmann
authors.affiliation: Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany.
authors.forename: Julia D
authors.initials: JD
authors.lastname: Suerth
authors.affiliation: Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany.
authors.forename: Tobias
authors.initials: T
authors.lastname: Maetzig
authors.affiliation: Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany.
authors.forename: Daniela
authors.initials: D
authors.lastname: Zychlinski
authors.affiliation: Dr. von Hauner Children's Hospital, Ludwig-Maximilians-University Munich, Munich, Germany.
authors.forename: Christoph
authors.initials: C
authors.lastname: Klein
authors.affiliation: Institute of Cell and Molecular Pathology, Hannover Medical School, Hannover, Germany.
authors.forename: Doris
authors.initials: D
authors.lastname: Steinemann
authors.affiliation: Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany.
authors.forename: Christopher
authors.initials: C
authors.lastname: Baum
authors.affiliation: Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany.
authors.forename: Juergen
authors.initials: J
authors.lastname: Bode
authors.affiliation: 1] Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany [2] Division of Hematology/Oncology, Children's Hospital Boston, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Axel
authors.initials: A
authors.lastname: Schambach
mesh_terms: D000818:Animals; D002454:Cell Differentiation / Q000235:genetics*; D064987:Cell- and Tissue-Based Therapy; D065150:Cellular Reprogramming; D019175:DNA Methylation; D004254:DNA Nucleotidyltransferases / Q000235:genetics*; D005822:Genetic Vectors; D057026:Induced Pluripotent Stem Cells; D016086:Lentivirus / Q000235:genetics; D051379:Mice; D020079:Terminal Repeat Sequences / Q000235:genetics*
chemical_list: D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase