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.
Highlights
The study and understanding of cell-fate decisions in early mammalian development can make use of cultures of Embryonic Stem Cells (ESCs), clonal populations derived from blastocysts which have the ability to self renew and differentiate into all cell types of an organism i.e., they are pluripotent[1,2]
As this had not been observed with mouse embryonic stem cells, we attempted to reproduce the behaviour observed with P19 Embryo Carcinoma (EC) cells using aggregates of mESCs19 and we report culture conditions that lead to their symmetry breaking and axial elongation
The technique described in this manuscript efficiently and reproducibly generates aggregates of mouse embryonic stem cells that display organized symmetry-breaking and elongation[19], combining both the hanging drop culture described by Marikawa et al.[14] and Embryoid Body (EB) formation
Summary
The study and understanding of cell-fate decisions in early mammalian development can make use of cultures of Embryonic Stem Cells (ESCs), clonal populations derived from blastocysts which have the ability to self renew and differentiate into all cell types of an organism i.e., they are pluripotent[1,2]. A report by Marikawa et al.[14], whilst working with aggregates of mouse P19 Embryo Carcinoma (EC) cells formed by the hanging-drop method, reported the emergence of elongated structures of mesodermal origin reminiscent of the elongations that are observed with exogastrulae in amphibian and sea urchin embryos and Keller explants[15,16,17,18] As this had not been observed with mouse embryonic stem cells (mESCs), we attempted to reproduce the behaviour observed with P19 EC cells using aggregates of mESCs19 and we report culture conditions that lead to their symmetry breaking and axial elongation. Three-dimensional aggregate culture offers a physical structure and signalling environment that may not be achievable by conventional means, leading to a new approach for the derivation of embryonic lineages in a spatially organized manner
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