Mammalian embryogenesis requires the coordination of embryonic and extra-embryonic tissues to enable implantation
into the uterus and post-implantation development to establish the body plan. Mouse embryonic stem cells (ESCs) are a
useful tool for studying pluripotent embryonic tissue in vitro. However, they cannot undertake correct embryogenesis
alone. Many attempts to model the early embryo in vitro involve the aggregation of ESCs into spheroids of variable size
and cell number that undertake germ-layer specification but fail to recapitulate the characteristic architecture and
arrangement of tissues of the early embryo. Here, we describe a protocol to generate the first embryo-like structures
by directing the assembly of mouse ESCs and extra-embryonic trophoblast stem cells (TSCs) in a 3D extracellular
matrix (ECM) into structures we call ‘polarized embryo-like structures’. By establishing the medium and culture
conditions needed to support the growth of both stem cell types simultaneously, embryonic architecture is generated
within 4 d of co-culture. This protocol can be performed by those proficient in standard ESC culture techniques and
can be used in developmental studies to investigate the interactions between embryonic and extra-embryonic tissues
during mammalian development.We are grateful to the Wellcome Trust for the Senior Research fellowship (grant no. 098287/Z/12/Z) and for a European Research Council grant (code: 669198) awarded to M.Z.-G. to fund this work. We are also grateful for the BBSRC DTP studentship that supports S.E.H. and to the Scientific and Technological Research Council of Turkey, which supports B.S.
