The progression from a fertilized oocyte to a fully developed newborn animal involves multiple cell divisions and developmental transitions, as initially pluripotent stem cells gradually commit to distinct cell lineages. A key developmental stage is the blastocyst stage â?? a time when embryonic (epiblast) and extra-embryonic (hypoblast) lineages are specified and segregate into distinct tissues. The epiblast, which gives rise to the embryo proper, is a structured pluripotent epithelium - a micro-scale tissue with tremendous developmental potential. The epiblast undergoes gastrulation to give rise to the three canonical germ layers of definitive endoderm, ectoderm, and mesoderm, which in turn generate all the tissues and organs in the developing embryo. Understanding this stage of development is thus of great importance to both developmental biology and tissue engineering.
The majority of our present understanding of mammalian embryology has been obtained in mouse, much of it using in-vivo-isolated blastocysts. This is a very powerful system, but it can also be labour intensive and expensive, with single experiments requiring several hundred embryos in some cases. There is therefore a significant need for in vitro model systems that will allow precise recapitulation of the earliest stages of embryogenesis.

To mimic early developmental stages, investigators have used embryoid bodies (EBs), which are aggregates of embryonic stem cells (ESCs) that form three dimensional structures that can be induced to differentiate into different cell lineages. While EBs have differences in geometry from the embryonic bilaminar disc, they have been widely and successfully used as a model system for this stage of development. While this work has primarily focused on murine cells, we have previously demonstrated that human ESCs exhibit similar potential, extending the applicability of this approach into other species where developmental studies have previously been limited for both ethical and practical reasons.
Subsequent induction of tissue-level
gastrulation behaviour has proved more challenging. This challenge is not unexpected, due to the well defined and oriented gradients of signalling molecules that are known to play a major role in embryonic axis formation and cell fate specification. There is thus a significant need for culture systems that permit recapitulation of the microenvironment of graded signals that is native to the bilaminar disc. I will present progress towards the development of microfluidic devices that will allow us to culture and manipulate individual colonies of pluripotent stem cells and provide a model system for the study of peri-implantation morphogenesis.

Fig. 1: Early mammalian embryogenesis. At the blastocyst stage, cells of the inner cell mass (ICM) (red, A) are proposed to form a mosaic with prospective hypoblast cells (green, B). After segregation according to cell type (C), the hypoblast cells lay down a basement membrane at the interface, and the ICM then polarizes and matures into the epiblast (D). We have recently demonstrated the capacity of human pluripotent stem cells to form analogous structures in vitro (E), consisting of an inner layer of columnar epithelial cells positive for the epiblast marker Oct4, surrounded by a laminin-containing basement membrane (F) and an outer layer of cells positive for the hypoblast marker GATA6 (G). Scale bar represents 50 microns.