Gastruloids generated without exogenous Wnt activation develop anterior neural tissues.

When stimulated with a pulse from an exogenous WNT pathway activator, small aggregates of mouse embryonic stem cells (ESCs) can undergo embryo-like axial morphogenesis and patterning along the three major body axes. However, these structures, called gastruloids, currently lack the anterior embryonic regions, such as those belonging to the brain. Here, we describe an approach to generate gastruloids that have a more complete antero-posterior development. We used hydrogel microwell arrays to promote the robust derivation of mouse ESCs into post-implantation epiblast-like (EPI) aggregates in a reproducible and scalable manner. These EPI aggregates break symmetry and axially elongate without external chemical stimulation. Inhibition of WNT signaling in early stages of development leads to the formation of gastruloids with anterior neural tissues. Thus, we provide a new tool to study the development of the mouse after implantation in vitro, especially the formation of anterior neural regions.

Bioengineered embryoids mimic post-implantation development in vitro.

The difficulty of studying post-implantation development in mammals has sparked a flurry of activity to develop in vitro models, termed embryoids, based on self-organizing pluripotent stem cells. Previous approaches to derive embryoids either lack the physiological morphology and signaling interactions, or are unconducive to model post-gastrulation development. Here, we report a bioengineering-inspired approach aimed at addressing this gap. We employ a high-throughput cell aggregation approach to simultaneously coax mouse embryonic stem cells into hundreds of uniform epiblast-like aggregates in a solid matrix-free manner. When co-cultured with mouse trophoblast stem cell aggregates, the resulting hybrid structures initiate gastrulation-like events and undergo axial morphogenesis to yield structures, termed EpiTS embryoids, with a pronounced anterior development, including brain-like regions. We identify the presence of an epithelium in EPI aggregates as the major determinant for the axial morphogenesis and anterior development seen in EpiTS embryoids. Our results demonstrate the potential of EpiTS embryoids to study peri-gastrulation development in vitro.