Abstract
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.
Highlights
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
The starting number of cells determined the size of the aggregate at 72 h of culture: an average of ~25 embryonic stem cells (ESCs) per microwell yielded aggregates with ~180 μm diameter, whereas seeding ~100 cells reached a diameter of ~230 μm (Fig. 1c)
These results show that polarized epithelial aggregates of defined size can be readily generated from ESCs using hydrogel microwell arrays, and confirmed that the provision of Matrigel is critical for their lumenization and epithelialization[16]
Summary
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. A challenge with ETS- and ETX-embryos is the reliance on the selforganizing activity of the cellular compartments that results in their stochastic occurrence; only ~20% of the starting aggregates will form patterned structures[11], which limits their broader applicability Their developmental potential is not yet clear, as there are no reports of their development beyond early gastrulation stages[11]. These pioneering in vitro models of early embryo development highlight the remarkable capacity of embryonic and extraembryonic cells to organize themselves but far have not yet been ideal to explore the fate and derivatives of cells in the emerging structures. Our approach enables the generation of various tissues in a stereotyped and highly scalable manner, with independent modulation of physical (e.g., size, epithelial architecture) and biological (e.g., provision of signaling molecules) properties of EPI and TSC aggregates, such as to systematically parse out the role of these parameters in promoting key steps in embryogenesis
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