The interplay between human germline specification and pluripotency factors
File(s)
Author(s)
Murison, Keir
Type
Thesis
Abstract
Pluripotency is regulated by transcription factors (TF) that maintain this cell state. In mouse, the same core pluripotency factors; Oct4, Nanog and Sox2 are expressed in the germline. The expression of these allows for the reversion of primordial germ cells (PGCs), founders of the gametes, into a pluripotent state in vitro.
Human primordial germ cells (PGCs) express OCT4 and NANOG but repress SOX2 and upregulate SOX17. Reports of similar conversion of human primordial germ cell-like cells (PGCLCs) or human germline tumours into a pluripotent state, in vivo or in vitro, require the downregulation of SOX17 and upregulation of SOX2. I hypothesised that overexpression of SOX2 within hPGCLCs might destabilise the germline network and trigger entry into a pluripotent state. I wanted to observe the effect of overexpression of other pluripotency related transcription factors (TFs), NANOG and KLF2, on germ cell-like cells.
To track entry into pluripotency, I utilised a cell line containing a reporter for endogenous SOX2 expression. A set of plasmids that overexpress the different pluripotency TFs of interest were transfected into the SOX2 reporter cells. These cell lines were then used to generate an in vitro cell type that resembles early human PGCs, called hPGC-like cells (hPGCLCs). The TFs could be activated and overexpressed at different points of the hPGCLC induction protocol and evidence of SOX2 re-activation could be observed.
I observed that SOX2 overexpression could not activate the endogenous SOX2 gene in any conditions studied, suggesting a dominant mechanism of repression probably mediated by BMP4 signalling. SOX2 could block entry into germline fate when overexpression was initiated at the point of hPGCLC induction but had no effect when triggered after induction. The other TF cell lines showed a failure to activate overexpression in the hPGCLC state, suggesting the inserted cassettes were silenced in hPGCLCs.
Human primordial germ cells (PGCs) express OCT4 and NANOG but repress SOX2 and upregulate SOX17. Reports of similar conversion of human primordial germ cell-like cells (PGCLCs) or human germline tumours into a pluripotent state, in vivo or in vitro, require the downregulation of SOX17 and upregulation of SOX2. I hypothesised that overexpression of SOX2 within hPGCLCs might destabilise the germline network and trigger entry into a pluripotent state. I wanted to observe the effect of overexpression of other pluripotency related transcription factors (TFs), NANOG and KLF2, on germ cell-like cells.
To track entry into pluripotency, I utilised a cell line containing a reporter for endogenous SOX2 expression. A set of plasmids that overexpress the different pluripotency TFs of interest were transfected into the SOX2 reporter cells. These cell lines were then used to generate an in vitro cell type that resembles early human PGCs, called hPGC-like cells (hPGCLCs). The TFs could be activated and overexpressed at different points of the hPGCLC induction protocol and evidence of SOX2 re-activation could be observed.
I observed that SOX2 overexpression could not activate the endogenous SOX2 gene in any conditions studied, suggesting a dominant mechanism of repression probably mediated by BMP4 signalling. SOX2 could block entry into germline fate when overexpression was initiated at the point of hPGCLC induction but had no effect when triggered after induction. The other TF cell lines showed a failure to activate overexpression in the hPGCLC state, suggesting the inserted cassettes were silenced in hPGCLCs.
Version
Open Access
Date Issued
2024-02
Date Awarded
2024-08
Copyright Statement
Creative Commons Attribution NonCommercial Licence
License URL
Advisor
Dion, Camille
Hajkova, Petra
Publisher Department
Institute of Clinical Sciences
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)