Biomechanical prerequisites for pluripotency

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Pluripotent stem cell lines are a valuable resource for biomedical and clinical research, having the ability to differentiate into any tissue of the body. Embryonic stem cells are derived from the epiblast of preimplantation mouse embryos. The epiblast (founder of the foetus) acquires pluripotency during its segregation from the primitive endoderm (which will produce the extra-embryonic yolk sac) in the inner cell mass of the blastocyst. This segregation is achieved by means of physical sorting from a 'salt and pepper' distribution into the two distinct tissues. This process has been observed by live imaging and characterised at the transcription level, but little is known about the biomechanical mechanisms by which epiblast and primitive endoderm tissues segregate, thereby establishing the pluripotent compartment of the embryo. To shed light on this important issue, we generated a list of candidates required for the structure of the cell's cytoskeleton from gene expression data (RNA sequencing) and protein expression (mass spectrometry). We will form our candidate list based upon representation in the epiblast and primitive endoderm and likelihood to influence cell biomechanics in culture. Armed with the initial candidate list, we will manipulate expression of the ~50 genes on this list by changing their expression artificially in a validated embryonic stem cell line. This line is unique in that it allows in vitro modelling of primitive endoderm segregation from epiblast by ectopic expression of a primitive endoderm-specific gene, thereby providing an excellent model of the inner cell mass of the early mouse embryo. By changing the expression of the candidates in our cell line, we will use biomechanical proxies such as colony forming assays and measurements of cell shape to narrow down our list. Then we will further elucidate the biomechanics of the cell line by measuring stiffness and tension of the cells, which are the main drivers of cell sorting. Consolidating the results of our experiments, we will select a shortlist of up to 10 candidate proteins. We will probe the effects of this biomechanical shortlist ex vivo using wild type preimplantation embryos. To do this, we will use two different types of experiments using our cell line with perturbations on candidate genes. First, we will force expression of the members of the candidate shortlist in one or more of the blastomeres (first embryonic cells) at the 2 or 4 cell stage. We expect to see effects on segregation to all three early embryonic lineages, including trophectoderm (that will form the placenta) using this assay. Second, to focus more on the establishment of plurioptency during epiblast/primitive endoderm sorting, we will use our cell line with relevant candidate perturbations using chimaeras by injection of donor cells into host wild type embryos at the 8 cell stage. Chimaeras that show the most significant phenotype will be transferred to wild type foster mice to allow development to progress until the lineages become more distinct (up to one week, within the first trimester of pregnancy). The output from this work will be a clearer understanding of the biomechanical mechanisms governing acquisition of true naïve pluripotency in the embryo that will enhance knowledge of early mammalian development and inform refinement of culture protocols for self-renewal or directed differentiation.