A multi-lineage in vitro mouse embryo model derived exclusively from mouse embryonic stem cells

The mouse embryo has been a vital model to study human embryonic development and congenital diseases. As the mouse embryo implants, it undergoes drastic morphological changes to form the egg cylinder consisting of the epiblast (EPI), extraembryonic ectoderm (ExE) and visceral endoderm (VE), which give rise to the embryo proper, placenta and yolk sac respectively. By using stem cells derived from these three fundamental lineages, many *in vitro* models have been developed to recapitulate various events of post-implantation development. Using only mouse embryonic stem cells (ESCs), the gastruloid model has been demonstrated to mimic basic body axis formation and aspects of gastrulation, somitogenesis, cardiogenesis, and neurulation. However, it fails to recapitulate the spatiotemporal interplay of signalling pathways between embryonic and extraembryonic tissues. Another approach is to combine ESCs with extraembryonic stem cells (trophoblast stem cells (TSCs) and extraembryonic endoderm stem cells (XEN cells)) in ETX embryos (embryo model composed of ESCs, TSCs and XEN cells) and iETX embryos (embryo model composed of ESCs, TSCs and induced XEN cells). While these integrated models have demonstrated greater developmental potentials, one remaining complication is that TSCs used in these models are maintained in undefined culture condition and are heterogenous in developmental state, which increase the difficulty and variability of embryoid formation. The central aim of this thesis is to develop an ESC-based *in vitro* embryo model that can reconstitute the three fundamental cell lineages of the post-implantation mouse embryo. It was shown that ESCs that transiently overexpressed Cdx2 upon doxycycline induction could effectively replace TSCs to form embryo-like structures. The resulting ‘EiTiX-embryoids’ underwent development from pre-gastrulation stages to neurulation stages, developing headfolds, a beating heart structure, and extraembryonic tissues, including a yolk sac and chorion. Further single-cell RNA sequencing analysis of individual embryoids showed a robust recapitulation of cell states in both embryonic and extraembryonic lineages, with little variation in the overall gene expression programme in these cell states. Together, this thesis presented the ‘EiTiX-embryoids’ as a novel ESC-based multi-lineage *in vitro* model of mouse post- implantation development to neurulation stages. This model combines the advantages of both existing ESC-based models and integrated models and it has reached an advanced developmental stage that is previously not achieved in ESC-based models. Lastly, this model can potentially be used in drug screening or as a novel disease model by using stem cell types with gene mutations.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2023

Author dc:creator

• Lau, Yui

Advisors dc:contributor.advisor

• Johnson, Randall
• Zernicka-Goetz, Magdalena

• embryogenesis
• embryoid
• in vitro model
• mouse embryonic stem cell

Licence

• https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/33b68c4c-3a12-4681-b2b2-fdf6fa308de6/download
• https://www.rioxx.net/licenses/all-rights-reserved/

Language dc:language

eng

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.102081

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/358423