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 Cathomen; Tom Struys; Catherine M. Verfaillie

doi:10.1016/j.stemcr.2015.09.004

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 journal › Article › peer-review

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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.

Original language	American English
Pages (from-to)	918-931
Number of pages	14
Journal	Stem Cell Reports
Volume	5
Issue number	5
DOIs	https://doi.org/10.1016/j.stemcr.2015.09.004
State	Published - 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.

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Ordovás, L., Boon, R., Pistoni, M., Chen, Y., Wolfs, E., Guo, W., Sambathkumar, R., Bobis-Wozowicz, S., Helsen, N., Vanhove, J., Berckmans, P., Cai, Q., Vanuytsel, K., Eggermont, K., Vanslembrouck, V., Schmidt, B. Z., Raitano, S., Van Den Bosch, L., Nahmias, Y., ... Verfaillie, C. M. (2015). Efficient recombinase-mediated cassette exchange in hPSCs to study the hepatocyte lineage reveals AAVS1 locus-mediated transgene inhibition. Stem Cell Reports, 5(5), 918-931. https://doi.org/10.1016/j.stemcr.2015.09.004

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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.",

author = "Laura Ordov{\'a}s and Ruben Boon and Mariaelena Pistoni and Yemiao Chen and Esther Wolfs and Wenting Guo and Rangarajan Sambathkumar and Sylwia Bobis-Wozowicz and Nicky Helsen and Jolien Vanhove and Pieter Berckmans and Qing Cai and Kim Vanuytsel and Kristel Eggermont and Veerle Vanslembrouck and Schmidt, {B{\'e}la Z.} and Susanna Raitano and {Van Den Bosch}, Ludo and Yaakov Nahmias and Toni Cathomen and Tom Struys and Verfaillie, {Catherine M.}",

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: {\textcopyright} 2015 The Authors.",

year = "2015",

month = nov,

day = "10",

doi = "10.1016/j.stemcr.2015.09.004",

language = "American English",

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Ordovás, L, Boon, R, Pistoni, M, Chen, Y, Wolfs, E, Guo, W, Sambathkumar, R, Bobis-Wozowicz, S, Helsen, N, Vanhove, J, Berckmans, P, Cai, Q, Vanuytsel, K, Eggermont, K, Vanslembrouck, V, Schmidt, BZ, Raitano, S, Van Den Bosch, L, Nahmias, Y, Cathomen, T, Struys, T & Verfaillie, CM 2015, 'Efficient recombinase-mediated cassette exchange in hPSCs to study the hepatocyte lineage reveals AAVS1 locus-mediated transgene inhibition', Stem Cell Reports, vol. 5, no. 5, pp. 918-931. https://doi.org/10.1016/j.stemcr.2015.09.004

TY - JOUR

T1 - Efficient recombinase-mediated cassette exchange in hPSCs to study the hepatocyte lineage reveals AAVS1 locus-mediated transgene inhibition

AU - Ordovás, Laura

AU - Boon, Ruben

AU - Pistoni, Mariaelena

AU - Chen, Yemiao

AU - Wolfs, Esther

AU - Guo, Wenting

AU - Sambathkumar, Rangarajan

AU - Bobis-Wozowicz, Sylwia

AU - Helsen, Nicky

AU - Vanhove, Jolien

AU - Berckmans, Pieter

AU - Cai, Qing

AU - Vanuytsel, Kim

AU - Eggermont, Kristel

AU - Vanslembrouck, Veerle

AU - Schmidt, Béla Z.

AU - Raitano, Susanna

AU - Van Den Bosch, Ludo

AU - Nahmias, Yaakov

AU - Cathomen, Toni

AU - Struys, Tom

AU - Verfaillie, Catherine M.

N1 - 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.

PY - 2015/11/10

Y1 - 2015/11/10

N2 - 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.

AB - 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.

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U2 - 10.1016/j.stemcr.2015.09.004

DO - 10.1016/j.stemcr.2015.09.004

M3 - Article

C2 - 26455413

AN - SCOPUS:84946714665

SN - 2213-6711

VL - 5

SP - 918

EP - 931

JO - Stem Cell Reports

JF - Stem Cell Reports

IS - 5

ER -

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

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