Generation of Transgene-Free Lung Disease-Specific Human Induced Pluripotent Stem Cells Using a Single Excisable Lentiviral Stem Cell Cassette §

Authors

  • Aba Somers,

    1. Boston University Pulmonary Center Boston University School of Medicine, Boston, Massachusetts, USA
    2. Department of Medicine, Boston University School of Medicine, Boston, Massachusetts, USA
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  • Jyh-Chang Jean,

    1. Boston University Pulmonary Center Boston University School of Medicine, Boston, Massachusetts, USA
    2. Department of Medicine, Boston University School of Medicine, Boston, Massachusetts, USA
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  • Cesar A. Sommer,

    1. Section of Gastroenterology, Department of Medicine, Boston University School of Medicine, Boston, Massachusetts, USA
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  • Amel Omari,

    1. Boston University Pulmonary Center Boston University School of Medicine, Boston, Massachusetts, USA
    2. Department of Medicine, Boston University School of Medicine, Boston, Massachusetts, USA
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  • Christopher C. Ford,

    1. Boston University Pulmonary Center Boston University School of Medicine, Boston, Massachusetts, USA
    2. Department of Medicine, Boston University School of Medicine, Boston, Massachusetts, USA
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  • Jason A. Mills,

    1. Center for Cellular and Molecular Therapeutics, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, USA
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  • Lei Ying,

    1. Center for Cellular and Molecular Therapeutics, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, USA
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  • Andreia Gianotti Sommer,

    1. Section of Gastroenterology, Department of Medicine, Boston University School of Medicine, Boston, Massachusetts, USA
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  • Jenny M. Jean,

    1. Boston University Pulmonary Center Boston University School of Medicine, Boston, Massachusetts, USA
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  • Brenden W. Smith,

    1. Section of Hematology and Oncology, Department of Medicine, Boston University School of Medicine, Boston, Massachusetts, USA
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  • Robert Lafyatis,

    1. Section of Rheumatology, Department of Medicine, Boston University School of Medicine, Boston, Massachusets, USA
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  • Marie-France Demierre,

    1. Department of Dermatology, Boston University School of Medicine, Boston, Massachusets, USA
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  • Daniel J. Weiss,

    1. Department of Medicine, University of Vermont College of Medicine, Burlington, Vermont, USA
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  • Deborah L. French,

    1. Center for Cellular and Molecular Therapeutics, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, USA
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  • Paul Gadue,

    1. Center for Cellular and Molecular Therapeutics, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, USA
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  • George J. Murphy,

    1. Section of Hematology and Oncology, Department of Medicine, Boston University School of Medicine, Boston, Massachusetts, USA
    2. Center for Regenerative Medicine (CReM), Boston University School of Medicine, Boston, Massachusets, USA
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  • Gustavo Mostoslavsky,

    Corresponding author
    1. Section of Gastroenterology, Department of Medicine, Boston University School of Medicine, Boston, Massachusetts, USA
    2. Center for Regenerative Medicine (CReM), Boston University School of Medicine, Boston, Massachusets, USA
    • Gustavo Mostoslavsky, Section of Gastroenterology, Department of Medicine, Boston University School of Medicine, 650 Albany Street X-513, Boston, Massachusetts 02118, USA

      Darrell N. Kotton, Boston University Pulmonary Center, and Department of Medicine, Boston University School of Medicine, 715 Albany St., R-304, Boston, Massachusetts 02118, USA

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    • Telephone: 617-638-6532; Fax: 617-6387785

  • Darrell N. Kotton

    Corresponding author
    1. Boston University Pulmonary Center Boston University School of Medicine, Boston, Massachusetts, USA
    2. Department of Medicine, Boston University School of Medicine, Boston, Massachusetts, USA
    3. Center for Regenerative Medicine (CReM), Boston University School of Medicine, Boston, Massachusets, USA
    • Gustavo Mostoslavsky, Section of Gastroenterology, Department of Medicine, Boston University School of Medicine, 650 Albany Street X-513, Boston, Massachusetts 02118, USA

      Darrell N. Kotton, Boston University Pulmonary Center, and Department of Medicine, Boston University School of Medicine, 715 Albany St., R-304, Boston, Massachusetts 02118, USA

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    • Telephone: 617-638-4860; Fax: 617-536-8063


  • Author contributions: A.S., J.C.J., C.A.S., C.C.F., J.A.M., and L.Y.: collection and assembly of data, data analysis and interpretation; A.O., A.G.S., and B.W.S.: collection and assembly of data; R.L., D.J.W., and M.F.D.: supply reagents; D.L.F. and P.G.: conception and design, data analysis and interpretation; G.J.M.: conception and design, collection and assembly of data, data analysis and interpretation, manuscript writing; G.M. and D.N.K.: conception and design, data analysis and interpretation, manuscript writing, final approval of manuscript. A.S., J.C.J., and C.A.S. contributed equally to this article.

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

  • §

    First published online in STEM CELLS EXPRESS August 16, 2010.

Abstract

The development of methods to achieve efficient reprogramming of human cells while avoiding the permanent presence of reprogramming transgenes represents a critical step toward the use of induced pluripotent stem cells (iPSC) for clinical purposes, such as disease modeling or reconstituting therapies. Although several methods exist for generating iPSC free of reprogramming transgenes from mouse cells or neonatal normal human tissues, a sufficiently efficient reprogramming system is still needed to achieve the widespread derivation of disease-specific iPSC from humans with inherited or degenerative diseases. Here, we report the use of a humanized version of a single lentiviral “stem cell cassette” vector to accomplish efficient reprogramming of normal or diseased skin fibroblasts obtained from humans of virtually any age. Simultaneous transfer of either three or four reprogramming factors into human target cells using this single vector allows derivation of human iPSC containing a single excisable viral integration that on removal generates human iPSC free of integrated transgenes. As a proof of principle, here we apply this strategy to generate >100 lung disease-specific iPSC lines from individuals with a variety of diseases affecting the epithelial, endothelial, or interstitial compartments of the lung, including cystic fibrosis, α-1 antitrypsin deficiency-related emphysema, scleroderma, and sickle-cell disease. Moreover, we demonstrate that human iPSC generated with this approach have the ability to robustly differentiate into definitive endoderm in vitro, the developmental precursor tissue of lung epithelia. STEM CELLS 2010;28:1728–1740

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