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Elife. 2018 Oct 9;7. pii: e36045. doi: 10.7554/eLife.36045.

Spatiotemporal mosaic self-patterning of pluripotent stem cells using CRISPR interference.

Author information

1
Gladstone Institute of Cardiovascular Disease, San Francisco, United States.
2
Developmental and Stem Cell Biology Graduate Program, University of California San Francisco, San Francisco, United States.
3
Graduate Program in Bioengineering, University of California Berkeley, University of California San Francisco, San Francisco, United States.
4
Department of Surgery, Center for Bioengineering and Tissue Regeneration, University of California, San Francisco, United States.
5
Department of Medicine, Division of Genomic Medicine, University of California, San Francisco, United States.
6
Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, United States.

Abstract

Morphogenesis involves interactions of asymmetric cell populations to form complex multicellular patterns and structures comprised of distinct cell types. However, current methods to model morphogenic events lack control over cell-type co-emergence and offer little capability to selectively perturb specific cell subpopulations. Our in vitro system interrogates cell-cell interactions and multicellular organization within human induced pluripotent stem cell (hiPSC) colonies. We examined effects of induced mosaic knockdown of molecular regulators of cortical tension (ROCK1) and cell-cell adhesion (CDH1) with CRISPR interference. Mosaic knockdown of ROCK1 or CDH1 resulted in differential patterning within hiPSC colonies due to cellular self-organization, while retaining an epithelial pluripotent phenotype. Knockdown induction stimulates a transient wave of differential gene expression within the mixed populations that stabilized in coordination with observed self-organization. Mosaic patterning enables genetic interrogation of emergent multicellular properties, which can facilitate better understanding of the molecular pathways that regulate symmetry-breaking during morphogenesis.

KEYWORDS:

bio-engineering; developmental biology; human; morphogenesis; pluripotent stem cells; regenerative medicine; stem cells

PMID:
30298816
PMCID:
PMC6177255
DOI:
10.7554/eLife.36045
[Indexed for MEDLINE]
Free PMC Article

Conflict of interest statement

AL, DJ, PS, JM, FM, VW No competing interests declared, MM Currently an employee of Tenaya Therapeutics. BC On the Scientific Advisory Boards of: CODA Therapeutics, making therapies related to pain relief; Cellogy Inc., a cell dynamics measurement company; and Scientist.com, a scientific services company. Also a founder of Tenaya Therapeutics (https://www.tenayatherapeutics.com/), a company focused on finding treatments for heart failure, including the use of CRISPR interference to interrogate genetic cardiomyopathies. Holds equity in Tenaya, and Tenaya provides research support for heart failure-related research. TM A consultant for Tenaya Therapeutics.

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