A ubiquitous chromatin opening element prevents transgene silencing in pluripotent stem cells and their differentiated progeny§

Authors

  • Nils Pfaff,

    1. REBIRTH Research Group Reprogramming, Hannover Medical School, Hannover, GermanyHannover Medical School, Hannover, Germany
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    • N.P. and N.L. contributed equally to this article.

  • Nico Lachmann,

    1. REBIRTH Research Group Reprogramming, Hannover Medical School, Hannover, GermanyHannover Medical School, Hannover, Germany
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    • N.P. and N.L. contributed equally to this article.

  • Mania Ackermann,

    1. REBIRTH Research Group Reprogramming, Hannover Medical School, Hannover, GermanyHannover Medical School, Hannover, Germany
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  • Saskia Kohlscheen,

    1. REBIRTH Research Group Reprogramming, Hannover Medical School, Hannover, GermanyHannover Medical School, Hannover, Germany
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  • Christian Brendel,

    1. Institute for Biomedical Research, Georg-Speyer-Haus, Frankfurt, Germany
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  • Tobias Maetzig,

    1. Institute of Experimental Hematology, Hannover Medical School, Hannover, Mariensee, Germany
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  • Heiner Niemann,

    1. REBIRTH Research Group Reprogramming, Hannover Medical School, Hannover, GermanyHannover Medical School, Hannover, Germany
    2. Institute of Farm Animal Genetics, Friedrich-Löffler-Institut, Mariensee, Germany
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  • Michael N. Antoniou,

    1. Gene Expression and Therapy Group, Department of Medical and Molecular Genetics, King's College London School of Medicine, Guy's Hospital, London, United Kingdom
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  • Manuel Grez,

    1. Institute for Biomedical Research, Georg-Speyer-Haus, Frankfurt, Germany
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  • Axel Schambach,

    1. Institute of Experimental Hematology, Hannover Medical School, Hannover, Mariensee, Germany
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  • Tobias Cantz,

    1. REBIRTH Research Group Stem Cell Biology, Hannover Medical School, Hannover, Germany
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  • Thomas Moritz

    Corresponding author
    1. REBIRTH Research Group Reprogramming, Hannover Medical School, Hannover, GermanyHannover Medical School, Hannover, Germany
    • Research Group Reprogramming, REBIRTH Cluster of Excellence, Hannover Medical School, Carl-Neuberg-Str.1, D-30625 Hannover, Germany
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    • Telephone: +49-511-532-5263; Fax: +49-511-532-5760


  • Author contributions: N.P. and N.L.: conception and design, collection and assembly of data, data analysis and interpretation, and manuscript writing; M.A.: collection and assembly of data and data analysis and interpretation; S.K.: collection and assembly of data; C.B., M.N.A., M.G., and A.S.: provision of study material; T. Maetzig: collection and assembly of data and provision of study material; H.N.: manuscript writing; T.C. and T. Moritz: conception and design and manuscript writing.

  • Disclosure of potential conflicts of interest is found at the end of this article.

  • §

    First published online in STEM CELLSEXPRESS January 2013, 2013.

Abstract

Methylation-induced gene silencing represents a major obstacle to efficient transgene expression in pluripotent cells and thereof derived tissues. As ubiquitous chromatin opening elements (UCOE) have been shown to prevent transgene silencing in cell lines and primary hematopoietic cells, we hypothesized a similar activity in pluripotent cells. This concept was investigated in the context of cytidine deaminase (CDD) gene transfer, an approach to render hematopoietic cells resistant to the chemotherapeutic agent Ara-C. When murine induced pluripotent stem cells (iPSC)/embryonic stem cells (ESCs) were transduced with self-inactivating lentiviral vectors using housekeeping (truncated elongation factor 1α; EFS) or viral (spleen focus-forming virus; SFFV) promoters, incorporation of an heterogeneous nuclear ribonucleoproteins A2 B1/chromobox protein homolog 3 locus-derived UCOE (A2UCOE) significantly increased transgene expression and Ara-C resistance and effectively prevented silencing of the SFFV-promoter. The EFS promoter showed relatively stable transgene expression in naïve iPSCs, but rapid transgene silencing was observed upon hematopoietic differentiation. When combined with the A2UCOE, however, the EFS promoter yielded stable transgene expression in 73% ± 6% of CD41+ hematopoietic progeny, markedly increased CDD expression levels, and significantly enhanced Ara-C resistance in clonogenic cells. Bisulfite sequencing revealed protection from differentiation-induced promoter CpG methylation to be associated with these effects. Similar transgene promoting activities of the A2UCOE were observed during murine neurogenic differentiation, in naïve human pluripotent cells, and during nondirected multilineage differentiation of these cells. Thus, our data provide strong evidence that UCOEs can efficiently prevent transgene silencing in iPS/ESCs and their differentiated progeny and thereby introduce a generalized concept to circumvent differentiation-induced transgene silencing during the generation of advanced iPSC/ESC-based gene and cell therapy products. STEM CELLS2013;31:488–499

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