Optimising human stem cell models to decipher signals and responses during organogenesis

The mammalian foetus is formed by a gradual process of tissue specification after the embryo has implanted in the uterus. Organs develop in an arrangement characteristic of each species through progressive differentiation from 3 distinct layers: ectoderm, endoderm and mesoderm, that separate physically and functionally during a process known as 'gastrulation'. Since this occurs within the mother, most studies have required removal of embryos, largely using the mouse as a model system. Gastrulating embryos can be cultured for several days outside the body, but this requires large numbers of mice (around 4 females for 20 embryos) and serum purified from blood of 4-5 rats. Embryonic stem cells (ESCs) are derived from preimplantation embryos and can be expanded indefinitely in culture whilst retaining capacity to differentiate into any tissue of the body. This is illustrated by injecting ESCs, usually after gene deletion or over-expression into preimplantation stage embryos and transferring the newly formed 'chimaeras' into foster mothers. Genetically-modified offspring are then selected for further breeding. To bypass the requirement for animals to address certain scientific questions, ESCs can be grown in 3D suspension culture using a simple protocol that allows them to undergo a process similar to gastrulation. These 3D 'gastruloids' can be guided to differentiate into recognisable tissues and rudimentary organs bearing strong physical and molecular resemblance to those of the embryo. Gastruloids are valuable, tractable tools, allowing researchers to reduce the number of embryos required for developmental studies. However, as mouse has limitations for human development, we and others have generated gastruloids from human ESCs. As human embryos cannot be used to study gastrulation due to ethical considerations, human gastruloids open a window to study human development that is otherwise inaccessible. Remarkably, the process that initiates symmetry breaking in gastruloids is spontaneous, but this makes it difficult to disentangle signals required for normal gastrulation and organ formation. Thus, we are faced with a major challenge to determine exactly how processes are initiated in a normal human embryo, which impedes our ability to uncover causes of embryonic abnormalities. Our project will tackle this problem by devising a system to control position, catchment area and duration of signalling cues to enhance understanding and enable controlled development of specific body parts in human gastruloids. We intend to focus on one internal organ, the gut, and an external structure, the limb bud. Rudimentary gut tubes can be induced in gastruloids showing some distinct regions approximating the foetal digestive tract. We will investigate the role of specialised 'neural crest cells' (NCCs), which are recruited to various developing tissues. In normal development NCCs emerge from the developing neural tube and migrate to produce various cell types, including those that form nerve cells in the developing gut. We will also inject NCCs to determine how these special neurons are recruited to the gut and whether they can contribute to its structural and functional development. This is particularly relevant for understanding defects such as Hirschsprung's disease. In addition, we will encapsulate developing gastruloids in customised gels to enable local application of substances known to induce formation and patterning of limb buds. Gastruloids generated from mouse ESCs have distinct regions in the flanks that express genes involved in limb formation. We will enhance limb bud development using human gastruloids and combine precisely positioned signalling factors with localised provision of NCC-derivatives that play a role in limb development. This project will demonstrate how human gastruloids provide a viable alternative to animal models that can be optimised to study gut and limb development and set the scene for future projects.

During early mammalian development, tissues and organs of the body emerge from rudiments or buds specified by local signals and reinforced by specific gene regulatory networks. Many of the essential molecular players in these processes have been identified using targeted gene disruption in (almost exclusively) animal models, but defining how genetic factors interact to specify, organise and regulate the various structures comprising the body in time and space has been challenging in vivo. Furthermore, there are limitations as to how well animal models mirror human development, and the inaccessibility (ethically and physically) of the human embryo to experimentation at these critical stages hampers our ability to examine this. In this project we will modify a novel 3Rs-compliant system, 'gastruloids', to examine the principles of how internal and external embryonic structures are formed, using the gut and limb bud - including neural crest cell (NCC) recruitment - as examples. Gastruloids are aggregates of embryonic stem cells that develop all three orthogonal embryonic axes in a spatially and temporally coordinated manner, mimicking early embryonic patterning events. Importantly, gastruloids develop bilateral focal regions molecularly resembling limb bud rudiments, and alteration of the protocol permits the development of Elongating Multi-Lineage Organoid (EMLO) gastruloids which mimic the formation of the embryonic gut. By using 3D hESC gastruloids/EMLOs, and collaborating with our industrial partner, Stem Cell Technologies, we will optimise a system directly relevant to study human tissue development that primarily uses only non-animal models, utilising quantitative molecular techniques (immunofluorescence, in situ hybridisation chain reaction, RNA sequencing), and non-animal derived matrices (hydrogels). This proof of principle will serve as a springboard for its wide dissemination and uptake.