Biomechanical prerequisites for pluripotency

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.

Although much is known about transciptional control of the specification of pluripotent epiblast (EPI) in the pre-implantation mouse embryo, little is known about biomechanical mechanisms, which have been proposed to be important for fate choice in embryogenesis. The specification of EPI and primitive endoderm (PrE) in the inner cell mass is achieved through an active segregation of the two lineages at the 64-128 cell stage. At this stage, we performed RNA sequencing to combine with an F-actin interactome produced using mass spectrometry to identify the cytoskeletal candidate factors correlated with specification of PrE and EPI. We will use an embryonic stem cell (ESC) line which forces ectopic expression of PrE genes in approximately 50% of the cells (leaving the rest as ESCs). This line produces ESCs and PrE like cells, and is therefore an excellent model of inner cell mass. The induced cell line sorts in vitro with the PrE like cells on the outside and ESCs on the inside. We will use knockdown and overexpression of candidate genes on both the ESCs and PrE like cells, and perform experiments to narrow down our biomechanical candidates. These experiments will consist of measurements of cell shape and defects in colony formation with perturbation of our candidates. With our compressed candidate list, we will measure cortical stiffness and tension using atomic force microscopy, as well as interfacial tension using cell doublet assays, to demonstrate biomechanical phenotypes and narrow down the candidate list. We will them perform mosaic experiments by by injecting synthetic mRNA and generate ESC chimaeras with our candidate list to deduce the importance of biomechanics in the specification of trophectoderm, PrE and EPI in the early mouse embryo. The results of our proposed research will help us to gain a better mechanistic understanding of morphogenesis, the importance of biomechanics in fate decisions of the early embryo, and the establishment of pluripotency.

The tools we develop and the knowledge acquired are anticipated to be applicable to improving the authenticity and reproducibility of pluripotent stem cell cultures for use in drug discovery and regenerative medicine. The general principles we aim to elucidate may be a paradigm for other stem cell systems, including human stem cells that could be tested with the same in vitro assays we develop. Furthermore, the cytoskeletal and biomechanical targets we will elucidate with this research may have possible impact in drug discovery, by showing how these biomechanical properties affect physiological processes such as cell adhesion, which is important in understanding cancer.

Proper identification the role of stem cells in developing new tissue, fundamental understanding of development and differentiation, and the availability of appropriate biotechnology promises to have an immense impact on medical practice. Our research will create a mechanistic approach to gaining a fundamental understanding of the establishment, maintenance and regulation of pluripotent stem cells, which could have an impact on medical practice by increasing efficiency, reliability and reducing treatment costs associated with regenerative medicine. This will enhance the quality of life on a national and international level.

Such a cutting-edge and interdisciplinary research project will further the skills and knowledge of researchers carrying out the research as well as all others (other group members, project students, colleagues) that we are in contact with. The impact will be a strengthening of problem solving and communication, as well as vocational and entrepreneurial skills, which will generally be useful in any later profession or project. Group members who gain direct experience with our innovative research methods will strengthen the skills base for biotechnology and health sciences in the UK.

In case patentable IP will arise from this project. Cambridge Enterprise, the University's commercialisation arm, will be consulted to evaluate our results for patentable IP and finding potential commercial partners beyond our existing network to ensure appropriate protection, exploitation and application of our research results.

During the course of this project, we will achieve the following project-specific objectives. First, we will elucidate biomechanical targets in early development, which could produce new possible targets for the pharmaceutical industry. Second, our in vitro assays will provide new means to investigate morphogenesis while reducing animal use. Third, we will explore patentable IP with Cambridge Enterprise. Once any appropriate IP is established, we will begin conversations with potential industrial partners to ultimately place our commercialisable technology appropriately into the marketplace.

We will also extensively communicate our results to the public, particularly regarding the potentialities of applying engineering and physical science approaches to biology, throughout the funding period. One aspect to this will be that we will design and deliver a specialised tour and lesson about the role of physics and new technology in cell biology for 14 - 19 year olds. We will also travel to scientific meetings to highlight the significance of our results. We will organise an International Symposium in Cambridge and explore through the Royal Society the potential for a workshop or conference on applying physical and biomechanical approaches to stem cell research.

Ultimately, we will impact human health by providing a greater understanding of pluripotency as the basis for regenerative medicine.