Generation of Aggregates of Mouse Embryonic Stem Cells that Show Symmetry Breaking, Polarization and Emergent Collective Behaviour In Vitro

Journal of Visualized Experiments : JoVE
Peter Baillie-JohnsonAlfonso Martinez Arias

Abstract

We have developed a protocol improving current Embryoid Body (EB) culture which allows the study of self-organization, symmetry breaking, axial elongation and cell fate specification using aggregates of mouse embryonic stem cells (mESCs) in suspension culture. Small numbers of mESCs are aggregated in basal medium for 48 hr in non-tissue-culture-treated, U-bottomed 96-well plates, after which they are competent to respond to experimental signals. Following treatment, these aggregates begin to show signs of polarized gene expression and gradually alter their morphology from a spherical mass of cells to an elongated, well organized structure in the absence of external asymmetry cues. These structures are not only able to display markers of the three germ layers, but actively display gastrulation-like movements, evidenced by a directional dislodgement of individual cells from the aggregate, which crucially occurs at one region of the elongated structure. This protocol provides a detailed method for the reproducible formation of these aggregates, their stimulation with signals such as Wnt/β-Catenin activation and BMP inhibition and their analysis by single time-point or time-lapse fluorescent microscopy. In addition, we describe mod...Continue Reading

References

Oct 11, 2016·Current Opinion in Cell Biology·Steffen WernerJochen C Rink
Apr 7, 2020·ELife·Alexandra SchauerCarl-Philipp Heisenberg
Aug 25, 2020·Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences·Matteo Rauzi
Apr 15, 2020·Life Science Alliance·Cristina AmeneiroMiguel Fidalgo

Related Concepts

Biological Markers
Embryo
Study
Decision Making
Entire Embryo
Malignant Neoplasm of Spinal Cord
Adherent Culture
Volar Plate
Science of Morphology
Aggregation

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