Characterisation of a reduced in-vitro 3D mouse model of post-implantation development

Early post-implantation development is a crucial step in mammalian embryogenesis and it is associated with high rate of pregnancies loss. Because embryo development at this stage occurs in the uterus, it is considered one of the most difficult developmental windows to study due to the inaccessibility of the embryo to observation. In the last few years, several stem cell-based models of post-implantation development have been generated, with a range of complexity that spans from relatively simple structures, composed of solely embryonic stem cells, to more complex ones, characterised by the co-culture of the former with extraembryonic stem cells. However, as the complexity of the system increases, the developmental efficiency decreases accordingly thus complex models of entire embryos are limited by efficiency of formation, despite mimicking many aspects of mammalian embryogenesis. In the present study I characterised a reduced model of early post-implantation development constituted by mouse embryonic stem cells and inducible extraembryonic endoderm stem cells. The resulting aggregates, called cysts, grew over the course of four days without providing neither any exogenous signalling cue nor three dimensional extracellular matrices for their development. Cysts underwent polarisation and lumenogenesis, resembling the epiblast at E5.5 at the pre-gastrula stage and comprised an epiblast-like compartment enveloped by a visceral endoderm-like layer. Initially, I performed optimisation experiments to determine the optimal number of cells required for efficient cyst formation, and then subsequent morphological analysis revealed possible associations between epiblast, lumen and overall structures dimensions. Timepoint characterisation demonstrated that cysts at 96 hours mimicked the epiblast at E5.5 with lumen formation occurring between 48 and 72 hours. Finally, targeted inhibition of TGF-β pathway revealed its role in regulation of epiblast pluripotency and overall cyst organisation. Together, my findings indicate that this reduced 3D mouse model of mouse post-implantation development represents an acceptable balance between efficiency and complexity and it can be successfully used to dissect molecular pathways important during early embryogenesis.