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Engineering the Spatiotemporal Mosaic Self-Patterning of Pluripotent Stem Cells

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Programmed Morphogenesis

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2258))

Abstract

Pluripotent stem cells (PSCs) possess the ability to self-organize into complex tissue-like structures; however, the genetic mechanisms and multicellular dynamics that direct such patterning are difficult to control. Here, we pair live imaging with controlled induction of gene knockdown by CRISPR interference (CRISPRi) to generate changes within subpopulations of human PSCs, allowing for control over organization and analysis of emergent behaviors. Specifically, we use forced aggregation of mixtures of cells with and without an inducible CRISPRi system to knockdown molecular regulators of tissue symmetry. We then track the resulting multicellular organization through fluorescence live imaging concurrent with the induction of knockdown. Overall, this technique allows for controlled initiation of symmetry breaking by CRISPRi to produce changes in cellular behavior that can be tracked over time within high-density pluripotent stem cell colonies.

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Author information

Authors and Affiliations

  1. Developmental and Stem Cell Biology PhD Program, University of California, San Francisco, CA, USA

    Ashley R. G. Libby

  2. Gladstone Institute of Cardiovascular Disease, Gladstone Institutes, San Francisco, CA, USA

    Ashley R. G. Libby, David A. Joy & Todd C. McDevitt

  3. UC Berkeley-UC San Francisco Graduate Program in Bioengineering, San Francisco, CA, USA

    David A. Joy

  4. Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, CA, USA

    Todd C. McDevitt

Authors
  1. Ashley R. G. Libby
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  2. David A. Joy
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  3. Todd C. McDevitt
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Corresponding author

Correspondence to Todd C. McDevitt .

Editor information

Editors and Affiliations

  1. Department of Pathology, Division of Experimental Pathology, School of Medicine, University of Pittsburgh, Pittsburgh Liver Research Center, University of Pittsburgh, Department of Bioengineering Swanson School of Engineering, University of Pittsburgh, McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, PA, USA

    Mo R. Ebrahimkhani

  2. Department of Bioengineering Swanson School of Engineering, Pittsburgh Liver Research Center, Department of Pathology, Division of Experimental Pathology, School of Medicine, University of Pittsburgh, Pittsburgh, PA, USA

    Joshua Hislop

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© 2021 The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

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Libby, A.R.G., Joy, D.A., McDevitt, T.C. (2021). Engineering the Spatiotemporal Mosaic Self-Patterning of Pluripotent Stem Cells. In: Ebrahimkhani, M.R., Hislop, J. (eds) Programmed Morphogenesis. Methods in Molecular Biology, vol 2258. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1174-6_8

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  • DOI: https://doi.org/10.1007/978-1-0716-1174-6_8

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1173-9

  • Online ISBN: 978-1-0716-1174-6

  • eBook Packages: Springer Protocols

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