BMP4-induced symmetry breaking in a 3D model of the human epiblast

Abstract We describe the protocol of generating a 3D stem-cell-based model of the human pre-gastrulation epiblast by culturing human embryonic stem cells in a mix of hydrogel and Matrigel. Much like the epiblast of an in vitro attached day-10 human embryo, this model is an epithelial sphere with a cavity at its center, it is expressing key pluripotency markers, and it displays apico-basal polarity. The 3D colonies can further be differentiated with morphogens and in the case of intermediate concentrations of BMP4, they break the anterior-posterior symmetry characterized by an asymmetric expression of a primitive streak marker and showing signs of epithelial to mesenchymal transition. The protocol described here is suitable for immunofluorescence staining and for live-cell imaging.