Towards an in vitro model of human hypoblast

It is widely known that the vast majority of pregnancies initiated following assisted conception programmes fail very early, at around the time of implantation. Although many of these failures can be attributed to incompatibility with the mother's uterus, around one third are caused by defects in the developing embryo. At the time of implantation, the embryo must consist of three tissues: the trophectoderm that will make the first connection with the uterus and give rise to the placenta; the hypoblast that is essential for specifying the anterior and posterior of the foetus and forming the yolk sac; and the epiblast that produces all the tissues of the foetus. In order for normal development to ensue, each early embryonic lineage must be appropriately and proportionately represented. Based on our observations, we hypothesise that failure to specify enough cells of either epiblast or hypoblast, or both, is likely to be a major problem for generating a viable pregnancy. However, the process by which appropriate allocation of these lineages is regulated is poorly understood. It is difficult to attain statistical power to answer questions about this process from embryos. Similarly, it is not possible to correlate apportionment of the lineages with eventual successful uterine implantation. Therefore, in order to understand how early embryonic lineages are allocated, it is essential to have model artificial embryos constructed from cell lines representing the early embryonic lineages. There are validated cell lines representing the epiblast and trophoblast, but not the hypoblast. We have used specially formulated supportive gels and a culture regime that can capture hypoblast cells from mouse embryos and keep them in an early embryonic state as they expand into cell lines. In this study, we will optimise the mechanical and chemical conditions specifically to generate self-renewing hypoblast cell lines from human embryos. Armed with cell lines representing all three embryonic lineages, we will use purpose-built 3D hydrogels to construct artificial embryos. With our model artificial embryos, in combination with the new endometrial organoids developed by our collaborator Margherita Turco, we can quantifiably test predictions concerning, for example, the number of cells of each lineage needed to initiate normal development, including implantation. Our study will provide valuable information on the requirements for specific factors for expanding the human hypoblast population that may enable improvement of culture regimes for assisted conception programmes.

In order to achieve successful embryonic implantation and pregnancy, the three early embryonic lineages - trophoblast, epiblast and hypoblast - must be allocated at the right location and in the right numbers. How this allocation is regulated, and how that relates to successful implantation, is poorly understood. The primary reason for this is that the regulation of early embryonic lineages is difficult to study in embryos, partly because of the practical difficulties of obtaining sufficient numbers for optimising multiple signalling conditions, and also because investigators must be able to carry the experiment through implantation to measure the outcome. Thus, in order to investigate the relationship between early lineage specification and implantation, it would be very useful to develop artificial embryos. Achieving this goal, however, is complicated by the fact that efforts to derive a cell line properly representing the nascent hypoblast have been mostly unsuccessful. In the proposed research, we will use our expertise in developmental biology and stem cell biophysics to optimise the biochemical and mechanical conditions to derive nascent hypoblast cell lines. We will first do this in mouse to learn what sorts of chemical and mechanical signalling requirements are necessary to harness a nascent hypoblast, then modify those conditions accordingly for nascent human hypoblast. We will then use these lines, combined with cell lines established by us and our collaborators representing nascent epiblast and trophoblast, to optimise the chemical and mechanical conditions to construct artificial embryos. We will then examine these artificial embryos and their interactions with endometrial organoids developed by our collaborator Margherita Turco to predict how early lineage allocation regulates implantation. With the proposed research, we ultimately hope to identify the prerequisites for successful implantation or failure.

Apart from academic beneficiaries, this work will be of interest and benefit to the general public because of the increasing use of assisted conception programmes for generating family units. There is still poor understanding of the problems occurring during the very early stages of pregnancy at around implantation. Our project will address this question at several levels. Firstly, the embryo culture experiments, with and without supplementary signalling molecules, followed by analysis of the number of cells per lineage will provide valuable information about the variability of proportions of the essential primary lineages between embryos. Although all patient information is anonymised, we can identify embryos from the same donors. Thus, we will be able to determine whether lineage-regulating phenotypes run in families. Secondly, the potential to generate artificial human embryos using derivative stem cell lines will allow formation of structures in which each lineage can be differentially labelled, providing an educational opportunity to capture the interest of the younger audience as well as determining the threshold cell numbers for successful implantation. Thirdly, there will be wide general interest in the potential to view the process of embryo implantation outside the body, especially if the composite lineages are differentially labelled. We will capture the process using timelapse microscopy and make the movies publicly available.
The fruits of our finding regarding the proportions of tissue, specifically hypoblast, needed for successful implantation may lead to suggestions for improvement of the currently used culture regimes during embryo expansion prior to transfer in IVF clinics, which would lead to a higher pregnancy success rate. The people benefitting from this knowledge will be the companies who provide the culture medium, the patients taking advantage of the treatment and clinicians, nurses and embryologists employed in assisted conception units.