Pluripotent embryonic stem cells (ESCs) can differentiate into any adult cell type; however, aggregates of these cells do not mimic embryonic architecture when grown in culture. To see whether mouse ESCs and their extraembryonic counterparts, trophoblast stem cells (TSCs), can recapitulate normal development, Harrison et al. combined ESCs and TSCs in an extracellular matrix culture (see the Perspective by Pera). The resultant “ETS-embryos” displayed considerable resemblance to normal embryos, even specifying mesoderm and primordial germ cells at the boundary between embryonic and extraembryonic compartments. These ETS-embryos are a genetically tractable tool for studying mammalian embryogenesis.

Structured Abstract

INTRODUCTION Early mammalian development requires the formation of embryonic and extraembryonic tissues and a highly coordinated partnership between them. This close partnership is a prerequisite for successful construction of embryo architecture, with the embryonic tissue generating cells of the embryo proper and the extraembryonic tissues, trophectoderm, and primitive endoderm forming the placenta and the yolk sac. Each of these components and the interactions between them are critical for embryonic development to birth. Embryonic stem cells (ESCs) in culture have the potential to participate in development when introduced into the early embryo. However, when cultured in vitro on their own, they do not recapitulate the spatial and temporal events of early embryogenesis.

RATIONALE We hypothesized that in order to faithfully model with stem cells the morphogenetic steps involved in mammalian embryogenesis, we would need to establish a developmental dialogue between ESCs and extraembryonic trophoblast stem cells (TSCs) in a three-dimensional (3D) extracellular matrix (ECM) scaffold, to potentially substitute for the basement membrane normally provided by the primitive endoderm.

RESULTS We combined embryonic and extraembryonic stem cells in vitro on such a 3D matrix and found that these cells were capable of self-assembly into a structure whose development and architecture were similar to that of the natural embryo, leading us to name them in vitro ESC and TSC stem cell–embryos (ETS-embryos). By building ETS-embryos from genetically modified stem cells and using specific inhibitors, we identify morphogenetic events and signaling pathways involved in these early developmental stages. Furthermore, we show that in vitro stem cell embryogenesis can be broken down into a sequence of key steps from implantation stage to germ layer specification. First is the self-organization of ESCs, which leads to polarization and lumenogenesis of ESC-derived embryonic compartment, followed by cavitation in the TSC-derived extraembryonic compartment. Second is the unification of embryonic and extraembryonic cavities into the equivalent of the embryo’s proamniotic cavity. Third is the requirement for a dialogue between embryonic and extraembryonic compartments, involving Nodal signaling, that builds characteristic embryo architecture. Fourth is the patterning of the embryonic compartment, revealed by the localized expression of mesoderm markers at the boundary between embryonic and extraembryonic compartments that, as in the natural embryo, is preceded by and dependent upon Wnt signaling. Fifth is the specification of a small cluster of PGC-like cells at the embryonic and extraembryonic boundary in a bone morphogenetic protein (BMP) signaling–dependent manner. Remarkably, such events in in vitro stem cell embryogenesis occur with very similar spatial and temporal dynamics to those taking place in natural embryogenesis.