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Assembly of embryonic and extraembryonic stem cells to mimic embryogenesis in vitro

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Science  14 Apr 2017:
Vol. 356, Issue 6334, eaal1810
DOI: 10.1126/science.aal1810

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In vitro embryogenesis

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.

Science, this issue p. eaal1810; see also p. 137

Structured Abstract


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.


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.


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.


In this work, we demonstrate that establishing a close cross-talk between embryonic and extraembryonic stem cells in a 3D ECM scaffold is necessary and sufficient for the self-assembly of a structure that recapitulates many of the key spatial and temporal steps of early mammalian embryogenesis. This in vitro ETS-embryo models embryonic architecture and patterning more accurately than has been possible before using ESCs alone. They also provide a simplified platform to dissect the physical, cellular, and molecular mechanisms mediating the critical cross-talk between embryonic and extraembryonic cells during development, and hence are a powerful tool for the future study of mammalian embryogenesis.

In vitro stem cell–embryos model mouse embryo development from implantation to gastrulation.

(Left) 3D rendered natural and ETS-embryos. Red, Oct4; cyan, Eomes. (Right) Parallel development of natural and ETS-embryos. Red, ESC/epiblast; blue, TSC/trophectoderm/extraembryonic ectoderm cells; green, primitive endoderm/visceral endoderm cells; dark green, distal/anterior visceral endoderm (DVE/AVE); beige, mesoderm cells; pale green, primordial germ cells; yellow line, basement membrane/ECM. Scale bars represent 30 μm.


Mammalian embryogenesis requires intricate interactions between embryonic and extraembryonic tissues to orchestrate and coordinate morphogenesis with changes in developmental potential. Here, we combined mouse embryonic stem cells (ESCs) and extraembryonic trophoblast stem cells (TSCs) in a three-dimensional scaffold to generate structures whose morphogenesis is markedly similar to that of natural embryos. By using genetically modified stem cells and specific inhibitors, we show that embryogenesis of ESC- and TSC-derived embryos—ETS-embryos—depends on cross-talk involving Nodal signaling. When ETS-embryos develop, they spontaneously initiate expression of mesoderm and primordial germ cell markers asymmetrically on the embryonic and extraembryonic border, in response to Wnt and BMP signaling. Our study demonstrates the ability of distinct stem cell types to self-assemble in vitro to generate embryos whose morphogenesis, architecture, and constituent cell types resemble those of natural embryos.

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