This award aims to use gastruloids cultured on synthetic hydrogels to replace embryos from genetically modified mice in studies of developmental interactions that require extracellular matrix.
Developmental events such as somitogenesis, a segmentation process that gives rise to vertebrae and skeletal muscles, rely on interactions with the extracellular matrix (ECM). These processes are generally studied in vivo using genetically modified mouse embryos isolated from pregnant mice that have been killed specifically for this purpose. When studying developmental processes in vitro, Matrigel is often used to provide matrix components, including proteins and sugars. Matrigel is derived from mouse tumours. Its components are not precisely defined and there is significant batch-to-batch variation. These limitations hamper the widespread use of in vitro models, such as gastruloids, 3D aggregates of embryonic stem cells, from being taken up to replace the use of mice for studying developmental events that require ECM. With NC3Rs funding, Professor Cathy Merry has previously developed synthetic hydrogels that support 3D culture for multiple cell types. These hydrogels are adaptable and various proteins and sugars can be added to replicate the components found in the ECM. Cathy will now work with Dr David Turner, a former NC3Rs-funded David Sainsbury Fellow, to combine her technology with the use of gastruloids.
The student will optimise the synthetic hydrogel for culturing gastruloids to enable in vitro studies of developmental events with precisely defined matrix components. They will use the hydrogels to determine the roles of specific proteins and sugars in developmental events and determine if extracellular matrix stiffness also plays a role in these events. The student will develop skills in 3D cell culture, hydrogel synthesis through collaborations with an industrial company, and microscopy imaging techniques working with Dr Jesse Veenvliet, Max Planck Institute, to demonstrate the utility and potential of the gastruloid method.
We envisage a time in the near future where the research community will advance humane, modern methodologies significantly reducing the need for animal experimentation and improving the quality and relevance of research. This studentship will develop an in vitro model of early embryogenesis by combining two novel technologies and then work with key figures in the developmental biology community to share the new methodology. In addition, recognising the 3Rs valley of death that limits widespread uptake of 3Rs relevant technologies, the student will also undertake a placement with a highly regarded peptide manufacturer, Pepceuticals, to learn how to manufacture, test and market products such as the hydrogels effectively.
Developed and optimised by Dr David Turner during his NC3Rs fellowship, gastruloids are aggregates of approximately 300 mouse embryonic stem cells which activate the same gene networks seen in a developing embryo and undergo patterning events such as polarised gene expression and gastrulation similar to that seen in a six to ten-day-old embryo. Gastruloids have been demonstrated to provide a replacement technique for genetically modified (GM) mouse embryos in the study of embryogenesis. The stem cells can be directly manipulated, removing the need for breeding GM mouse lines in addition to avoiding the need to kill pregnant mice for embryo collection. However, studying later developmental events such as somitogenesis requires additional support derived from interactions with the extracellular matrix. Currently, that matrix is usually provided by Matrigel, a commercial preparation from mouse tumour extract, with significant batch-to-batch variation and complexity that makes it difficult to unpick complex biological questions. Matrigel can mask disease-specific cell-matrix interactions which are of significance in modelling disorders where these are an important part of the disease pathology and resistance to therapy.
To solve the Matrigel problem, the Merry lab have developed synthetic peptide hydrogels (SPH). We have used them to create in vitro models of breast cancer and demonstrated they support 3D culture for multiple other cell types, including embryonic stem cells. The SPH technology is reproducible and adaptable and has been successfully shared with other groups. The SPH provide a blank slate with which to test the role of matrix proteins and glycans as well as to evaluate the role of matrix stiffness. To investigate that aspect further, we’ve also teamed up with an expert in optics, Dr Amanda Wright, with whom we have developed a new microscope that allows us to understand how cell clusters interact with their local matrix at the cellular level. The high levels of reproducibility possible using the gastruloid and SPH technologies will enable us to build a detailed map of the local mechanical forces that impact on early developmental decisions and will showcase the new model system to encourage uptake.
The student will work with an engaged and focused supervisory team that will additionally provide access to their extended networks of collaborators. We’ve previously found that working closely with a few, key groups to understand what they need from a 3Rs compliant technology helps when trying to encourage them to move away from using animals or animal-derived materials. We’ve therefore established a strong collaboration with Dr Jesse Veenvliet, a group leader at the Max Plank Institute of Molecular Cell Biology and Genetics in Dresden. The student will be in good communication with the group during their PhD and will undertake a working visit to access additional imaging tools. During the visit, the student will also demonstrate the combined gastruloid/SPH technology to an institute which is at the forefront of research in this area and where we can gain an excellent understanding of how to best present an alternative to Matrigel to this community.