Efficient recombinase-mediated cassette exchange in hPSCs to study the hepatocyte lineage reveals AAVS1 locus-mediated transgene inhibition

Laura Ordovás*, Ruben Boon, Mariaelena Pistoni, Yemiao Chen, Esther Wolfs, Wenting Guo, Rangarajan Sambathkumar, Sylwia Bobis-Wozowicz, Nicky Helsen, Jolien Vanhove, Pieter Berckmans, Qing Cai, Kim Vanuytsel, Kristel Eggermont, Veerle Vanslembrouck, Béla Z. Schmidt, Susanna Raitano, Ludo Van Den Bosch, Yaakov Nahmias, Toni CathomenTom Struys, Catherine M. Verfaillie

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

90 Scopus citations


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.

Original languageAmerican English
Pages (from-to)918-931
Number of pages14
JournalStem Cell Reports
Issue number5
StatePublished - 10 Nov 2015

Bibliographical note

Funding Information:
L.O. was funded by IWT/OZM/090838, IACS BPAMER3/08/04, and Government of Aragon FMI048/08; M.P. by FWO 1288714N; and R.S. by the Dutch Diabetes Foundation. R.B., N.H., J.V., K.C., and Q.C. were supported by IWT fellowships SB-121393, SB-121396, SB-101230, SB-091228, and SB-093228, respectively. B.Z.S. was financed by FP7-PEOPLE-2011-IIF (Proposal No. 298785) and W.G. by the China Scholarship Council. S.B.-W. and T.C. were supported by the FP7-HEMIBIO (266777). Funding to C.M.V. was from FWO G.0667.07 and G.0975.11; KU Leuven (EIW-B4855-EF/05/11, ETH-C1900-PF, EME-C2161-GOA/11/012), IWT-HEPSTEM, BELSPO-IUAP-DEVREPAIR, FP7-HEMIBIO (266777). We thank Prof. P. Collas and Dr. B. Izzi for their discussions related to the DNA methylation studies; Dr. D. Franckaert, Dr. S. Schlenner, and Dr. A. Van Nieuwenhuijze for their availability to operate the sorter as well as M. Welters and R. Van Rossom for technical assistance.

Publisher Copyright:
© 2015 The Authors.


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