Generation of human embryonic stem cell-derived mesoderm and cardiac cells using size-specified aggregates in an oxygen-controlled bioreactor

Authors

  • Sylvia Niebruegge,

    1. Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Ontario, Canada; telephone: 416-978-8888; fax: 416-978-2666
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  • Céline L. Bauwens,

    1. Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Ontario, Canada; telephone: 416-978-8888; fax: 416-978-2666
    2. Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario, Canada
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  • Raheem Peerani,

    1. Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Ontario, Canada; telephone: 416-978-8888; fax: 416-978-2666
    2. Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario, Canada
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  • Nimalan Thavandiran,

    1. Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Ontario, Canada; telephone: 416-978-8888; fax: 416-978-2666
    2. Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario, Canada
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  • Stephane Masse,

    1. Division of Cardiology, Toronto General Hospital, Toronto, Ontario, Canada
    2. The Toby Hull Cardiac Fibrillation Management Laboratory, Toronto General Hospital, Toronto, Ontario, Canada
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  • Elias Sevaptisidis,

    1. Division of Cardiology, Toronto General Hospital, Toronto, Ontario, Canada
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  • Kumar Nanthakumar,

    1. Division of Cardiology, Toronto General Hospital, Toronto, Ontario, Canada
    2. The Toby Hull Cardiac Fibrillation Management Laboratory, Toronto General Hospital, Toronto, Ontario, Canada
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  • Kim Woodhouse,

    1. Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario, Canada
    2. Faculty of Applied Science, Queen's University, Toronto, Ontario, Canada
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  • Mansoor Husain,

    1. Division of Cardiology, Toronto General Hospital, Toronto, Ontario, Canada
    2. Heart & Stroke Richard Lewar Centre of Excellence in Cardiovascular Research, University of Toronto, Toronto, Ontario, Canada
    3. McEwen Centre for Regenerative Medicine, Toronto General Hospital, Toronto, Ontario, Canada
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  • Eugenia Kumacheva,

    1. Department of Chemistry, University of Toronto, Toronto, Ontario, Canada
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  • Peter W. Zandstra

    Corresponding author
    1. Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Ontario, Canada; telephone: 416-978-8888; fax: 416-978-2666
    2. Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario, Canada
    3. Heart & Stroke Richard Lewar Centre of Excellence in Cardiovascular Research, University of Toronto, Toronto, Ontario, Canada
    4. McEwen Centre for Regenerative Medicine, Toronto General Hospital, Toronto, Ontario, Canada
    • Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Ontario, Canada; telephone: 416-978-8888; fax: 416-978-2666.
    Search for more papers by this author

Abstract

The ability to generate human pluripotent stem cell-derived cell types at sufficiently high numbers and in a reproducible manner is fundamental for clinical and biopharmaceutical applications. Current experimental methods for the differentiation of pluripotent cells such as human embryonic stem cells (hESC) rely on the generation of heterogeneous aggregates of cells, also called “embryoid bodies” (EBs), in small scale static culture. These protocols are typically (1) not scalable, (2) result in a wide range of EB sizes and (3) expose cells to fluctuations in physicochemical parameters. With the goal of establishing a robust bioprocess we first screened different scalable suspension systems for their ability to support the growth and differentiation of hESCs. Next homogeneity of initial cell aggregates was improved by employing a micro-printing strategy to generate large numbers of size-specified hESC aggregates. Finally, these technologies were integrated into a fully controlled bioreactor system and the impact of oxygen concentration was investigated. Our results demonstrate the beneficial effects of stirred bioreactor culture, aggregate size-control and hypoxia (4% oxygen tension) on both cell growth and cell differentiation towards cardiomyocytes. QRT-PCR data for markers such as Brachyury, LIM domain homeobox gene Isl-1, Troponin T and Myosin Light Chain 2v, as well as immunohistochemistry and functional analysis by response to chronotropic agents, documented the impact of these parameters on cardiac differentiation. This study provides an important foundation towards the robust generation of clinically relevant numbers of hESC derived cells. Biotechnol. Bioeng. 2009;102: 493–507. © 2008 Wiley Periodicals, Inc.

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