Cell fate regulation during gastrulation in humans and pigs

To the renowned embryologist Lewis Wolpert is attributed the quote: "It is not birth, marriage or death, but gastrulation which is truly the most important time in your life".
Gastrulation is the period of embryo development (around the second week of pregnancy in humans) when the major cell precursors that will form the organs in our body are determined. It is a critical time because if such development is disrupted, it can lead to irreversible abnormalities or have long term detrimental consequences for health in later life.
How the gastrulation process occurs in humans is very poorly understood because human embryos cannot be directly studied during this period for ethical reasons. Mouse embryos have served as good models for understanding general principles of mammalian development, but we also know that mice and humans are very different in their development. Indeed, some of the key developmental differences correlate with distinct embryo morphology in mice and humans. Mouse embryos develop as a conical structure in which interactions with tissues contributing to the placenta are very different from other large mammals. Humans develop flat embryos, which establish different relationships with placental tissues. We have studied the significance of these differences in detail in pig embryos, which also develop as flat discs like humans. Our research showed that the genes that regulate the formation of sperm and egg precursor cells are different between pigs and mice. Importantly, we also showed that the mechanism used by pig is also used in human and other primate embryos. This landmark study showed for the first time that the molecular identity of a cell is different between mice and other larger mammals. Furthermore, it indicated that pig embryos can be used as a model for non-rodents mammals and be used to help understand human development.
Further research is needed to find out whether differences also occur during the formation of other cells and organs, such as the muscle, liver, gut and pancreas. Such new understanding will ultimately help inform methods for driving human stem cells towards specific cell types with therapeutic potential.
The first objective of the research is to determine the genetic program of different cell precursors during gastrulation in pig embryos. Using single cell molecular analysis we will build a map of how cells become restricted towards different cellular identities. This data will be then compared with available data from mouse and human stem cell differentiation studies that will allow us to establish similarities in developmental mechanisms between species.
Next, we will validate these findings using new genome editing technologies to disrupt protein function in embryos and determine the morphology of mutant embryos. We will determine cell identity in mutant embryos and establish the roles played by these key proteins during this critical period of development.
The final objective of the research is to determine the developmental pathway of human cells in pig/human chimeric embryos. Embryos generated from two species, known as chimeras, are excellent tools for assessing cell developmental potential in vivo. Here we will test the capacity of newly establish hESC and other progenitors combined with chemical methods to prevent cell death to enhance chimera integration. We will use labelling technologies to track cell development and determine cell fate in pig embryos.
Understanding how pig/human chimeras develop will be an important step towards generating human organs in large animals for xenotransplantation. A better understanding of the biological barriers and technical limitations of these approaches will improve the safety and efficacy of these new transplantation technologies.

The mechanisms of gastrulation in non-rodent mammals are very poorly understood because traditionally, findings in mice were considered representative of all mammals. Recent studies have shown that pre-implantation mouse embryos have distinct developmental programs from those of humans, non-human primates and pigs. Less is known about gastrulation differences; however recent evidence from our laboratory shows that the molecular program of germ cell development in humans and pigs is shared, and is different from that in mice. We hypothesize that the mechanisms of human and pig gastrulation are largely conserved, and that the pig is an alternative model system relevant to human gastrulation. We will extend the observations made in the pig germline to other somatic cell lineages and use pig/human embryonic chimeras to assess human stem cell differentiation potential in vivo.

Objectives of the proposal:

Objective 1: We will delineate the segregation of somatic lineages (ecto-, meso-, and endoderm) in the pig embryo using scRNA seq. of pig embryos from different peri-gastrulation stages. We will identify key genes involved in lineage decisions during gastrulation and use bioinformatic analysis to reconstruct the progressive decisions made during gastrulation. These findings will be compared to the developmental gene signatures of primate and mouse embryos available in public databases.

Objective 2: We will perform functional evaluation of the roles of identified key transcription factors during gastrulation. The functions of key transcription factors identified by scRNA-Seq will be validated using gene editing of pig zygotes, followed by embryo transfer and retrieval of embryos at the onset of gastrulation.

Objective 3: We will determine human PSC and endoderm progenitors differentiation capacity in pig chimeras. We will use interspecific chimeras to test the differentiation capacity of hPSC and endoderm progenitors in pig embryos in vivo.

Below is a list of the major stakeholders benefiting from this research:

Biomedical Research (Stakeholders: Academic and Industrial R&D): Developing improved methodologies for the generation of specific cell types from human embryonic stem cells will greatly improve the translational applications of these technologies in human regenerative medicine. Great demand exists for improving such technologies in the face of the potential solutions they may offer for treating degenerating diseases of ageing populations. Current limitations of these technologies are the low proportion of adult mature cell types produced in vitro. Our research will generate new understanding of how cells differentiate in vivo which will inform more robust differentiation approaches for obtaining desired functional cell types suitable for transplantation, for toxicological screening and disease modelling.

Human lifelong health and wellbeing (stakeholders: individuals/society): The increasing demand for organs in the UK (rising at 4% a year) indicates that there is critical need for alternative sources of organs for transplantation. Using genetic modification, pigs could be engineered to carry humanized organs, which may be suitable sources of organs for xenotransplantation. Pigs and human share many physiological and anatomical features. Pig hearts and kidneys have a similar size to a human equivalents. Pig corneas and pancreas can be made human compatible by genetic engineering. In addition, the technology developed here will also enable the generation of whole human organs in pigs, by using a combination of gene ablation plus embryo complementation with human cells to generate pig/human interspecies chimeras. These new technologies will bring the possibility of generating safe organs for transplantation to humans a step closer.

Ethics/policy making (Stakeholders: government/society): The new knowledge generated in this research will underpin a pathway to the development on novel medical approaches, which will have transformative impact in regenerative medicine and cell therapy. The possibility of generating human organs in interspecies chimeras raises ethical concerns. Therefore a thorough benefit/risk analysis needs to be carried out to determine the value of these new technologies. The societal benefits of developing alternative sources of organs need to outweigh the ethical concerns raised to have the desired impact in solving pressing medical problems, such as the lack of sufficient organs for transplantation and our increased needs for replacement organs to increase life expectancy. Policy makers and government agencies will benefit from these new findings and will use them to inform their decisions on the regulation of these new technological developments.