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Stem cells are undifferentiated cells that divide and produce different types of cells. Ordinarily, cells that have differentiated into a specific cell type are post-mitotic—that is, they no longer divide. However, scientists have found a way to reprogram these mature cells so that they “de-differentiate” and return to an unspecialized, proliferative state. These cells are also pluripotent like embryonic stem cells—able to produce all cell types—and are therefore...
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Stencil Micropatterning of Human Pluripotent Stem Cells for Probing Spatial Organization of Differentiation Fates
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Engineering the Spatiotemporal Mosaic Self-Patterning of Pluripotent Stem Cells.

Ashley R G Libby1,2, David A Joy2,3, Todd C McDevitt4,5

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

Methods in Molecular Biology (Clifton, N.J.)
|December 19, 2020
PubMed
Summary

Researchers developed a new method using CRISPR interference (CRISPRi) and live imaging to control how human pluripotent stem cells (PSCs) organize. This technique allows for studying gene function in self-organizing cell populations.

Keywords:
CRISPR interferenceCell trackingForced aggregationLive imagingMorphogenesisPluripotent stem cells

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Area of Science:

  • Stem cell biology
  • Developmental biology
  • Gene editing technologies

Background:

  • Pluripotent stem cells (PSCs) exhibit self-organization into complex structures.
  • Controlling genetic mechanisms and multicellular dynamics in PSCs is challenging.
  • Understanding tissue patterning requires advanced analytical tools.

Purpose of the Study:

  • To develop a method for controlling organization and analyzing emergent behaviors in human PSCs.
  • To investigate the role of specific genes in tissue symmetry and patterning.
  • To enable precise manipulation of cellular subpopulations within PSC colonies.

Main Methods:

  • Utilizing inducible CRISPR interference (CRISPRi) for controlled gene knockdown in subpopulations of human PSCs.
  • Employing forced aggregation of mixed cell populations (CRISPRi-inducible and control).
  • Performing fluorescence live imaging to track multicellular organization and cellular behaviors over time.

Main Results:

  • Demonstrated controlled initiation of symmetry breaking through targeted gene knockdown.
  • Successfully generated changes within specific cell subpopulations to influence overall colony organization.
  • Enabled real-time tracking of emergent behaviors in response to genetic perturbations.

Conclusions:

  • The combined approach of CRISPRi and live imaging offers unprecedented control over PSC self-organization.
  • This technique facilitates the study of genetic regulators of tissue patterning and emergent multicellular dynamics.
  • Provides a powerful platform for dissecting the mechanisms underlying stem cell self-organization.