title: Generation of Aggregates of Mouse Embryonic Stem Cells that Show Symmetry Breaking, Polarization and Emergent Collective Behaviour In Vitro.
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 modifications to current whole-mount mouse embryo staining procedures for immunocytochemical analysis of specific markers within fixed aggregates. The changes in morphology, gene expression and length of the aggregates can be quantitatively measured, providing information on how signals can alter axial fates. It is envisaged that this system can be applied both to the study of early developmental events such as axial development and organization, and more broadly, the processes of self-organization and cellular decision-making. It may also provide a suitable niche for the generation of cell types present in the embryo that are unobtainable from conventional adherent culture such as spinal cord and motor neurones.
authors.affiliation: Department of Genetics, University of Cambridge.
authors.forename: Peter
authors.initials: P
authors.lastname: Baillie-Johnson
authors.affiliation: Department of Genetics, University of Cambridge; Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences.
authors.forename: Susanne Carina
authors.initials: SC
authors.lastname: van den Brink
authors.affiliation: Department of Genetics, University of Cambridge.
authors.forename: Tina
authors.initials: T
authors.lastname: Balayo
authors.affiliation: Department of Genetics, University of Cambridge.
authors.forename: David Andrew
authors.initials: DA
authors.lastname: Turner
authors.affiliation: Department of Genetics, University of Cambridge; ama11@hermes.cam.ac.uk.
authors.forename: Alfonso
authors.initials: A
authors.lastname: Martinez Arias