A study into the biology of mESCs exiting naïve pluripotency and technology development for subcellular proteomics

Developmental biologists have been fascinated by different aspects of embryology for decades. One of those aspects is pluripotency, the ability of embryonic cells of certain developmental stages to divide indefinitely without the loss of their potential to differentiate into derivatives of the three primary germ layers. Pluripotency has been captured in vitro by the establishment of cell culture conditions that enable scientists to maintain and propagate Pluripotent Stem Cells (PSCs) such as Embryonic Stem Cells (ESCs) in the dish. Mouse embryonic pluripotency, specifically, is represented in vitro by naïve ESCs, which resemble the pre-implantation mouse embryo, and primed Epiblast Stem Cells (EpiSCs), which are most similar to the post-implantation mouse embryo. In recent years, the exit of mESCs from naive pluripotency has been partially characterised, and several protocols have been developed that can generate either transient or stable cellular states which are intermediate to mESC and EpiSC pluripotency, such as Epiblast-Like Cells (EpiLCs). Studies that have examined mESC pluripotency and early differentiation dynamics have suggested that mESCs transition towards lineage specification through a transient state of primed pluripotency that resembles that of EpiSC pluripotency. However, this hypothesis has not been methodically interrogated. In this PhD thesis, I study this biological question by performing an extensive comparison of mESCs at the early stages of the default program of neurectodermal differentiation versus primed EpiSCs as well as pre-primed EpiLCs. I include in this comparison both low-throughput molecular and functional assays but also a shotgun proteomics based analysis of the global proteomes of differentiating mESCs versus EpiSCs. I demonstrate that, although mESCs at the beginning of their differentiation process share common characteristics with both naïve and primed PSCs, they represent a cellular state which is distinct to those of EpiLCs and EpiSCs. Lastly, in the last chapter of my PhD thesis, I present my work on the development of a new method for the analysis of protein subcellular localisation, Localisation of Organelle Proteins by Isotope Tagging after Differential ultraCentrifugation (LOPIT-DC). LOPIT-DC can be employed in the future to expand the comparison between distinct states of pluripotency towards an additional dimension, specifically by interrogating the role of protein subcellular localisation dynamics in regulating pluripotency state transitions.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2025

Author dc:creator

• Geladaki, Aikaterini

Advisor dc:contributor.advisor

• Lilley, Kathryn

• pluripotency
• subcellular proteomics

Licence

• https://www.repository.cam.ac.uk/bitstreams/9797fac0-0a1d-4946-a6cb-929232074460/download
• https://www.nationalarchives.gov.uk/doc/open-government-licence/version/3/

Language dc:language

eng

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.120718

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/388320