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29 maart 2021, Contains fulltext : 235970.pdf (Publisher’s version ) (Open Access)

Radboud University, 11 oktober 2021, Promotor : Stunnenberg, H.G. Co-promotor : Marks, H., Contains fulltext : 237502.pdf (Publisher’s version ) (Open Access)

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Contains fulltext : 196239.pdf (publisher's version ) (Open Access), During early mammalian development, transient pools of pluripotent cells emerge that can be immortalised upon stem cell derivation. The pluripotent state, 'naive' or 'primed', depends on the embryonic stage and derivation conditions used. Here we analyse the temporal gene expression patterns of mouse, cattle and porcine embryos at stages that harbour different types of pluripotent cells. We document conserved and divergent traits in gene expression, and identify predictor genes shared across the species that are associated with pluripotent states in vivo and in vitro Amongst these are the pluripotency-linked genes Klf4 and Lin28b The novel genes discovered include naive- (Spic, Scpep1 and Gjb5) and primed-associated (Sema6a and Jakmip2) genes as well as naive to primed transition genes (Dusp6 and Trip6). Both Gjb5 and Dusp6 play a role in pluripotency since their knockdown results in differentiation and downregulation of key pluripotency genes. Our interspecies comparison revealed new insights of pluripotency, pluripotent stem cell identity and a new molecular criterion for distinguishing between pluripotent states in various species, including human.

Contains fulltext : 197905.pdf (publisher's version ) (Open Access)

Contains fulltext : 168894.pdf (preprint version ) (Open Access), Mouse embryonic stem (ES) cells are locked into self-renewal by shielding from inductive cues. Release from this ground state in minimal conditions offers a system for delineating developmental progression from naive pluripotency. Here we examined the initial transition process. The ES cell population behaves asynchronously. We therefore exploited a short-half-life Rex1::GFP reporter to isolate cells either side of exit from naive status. Extinction of ES cell identity in single cells is acute. It occurs only after near-complete elimination of naïve pluripotency factors, but precedes appearance of lineage specification markers. Cells newly departed from the ES cell state display features of early post-implantation epiblast and are distinct from primed epiblast. They also exhibit a genome-wide increase in DNA methylation, intermediate between early and late epiblast. These findings are consistent with the proposition that naive cells transition to a distinct formative phase of pluripotency preparatory to lineage priming.

Free-electron lasers are used to generate laser radiation using an accelerated electron beam. In this paper, an electrostatic accelerator was used with a thermionic cathode. Lasing pulses of the order of tens of microseconds were generated by passing the current pulses through a wiggler and resonator combination. The change in lasing power as a result of postsaturation variation of the energy of the electron beam during the lasing period is presented in this paper in the form of experimental measurements and simulations. In the experiments, the initial electron beam energy was similar to 1.4 MeV and the current was 1.13 A, whilst the lasing frequency was always close to 102 GHz. It is shown that postsaturation, positively ramping the beam energy increases the radiation extraction efficiency by around 50%. Whilst a falling beam energy results in a reduction in extraction efficiency.

Mouse embryonic stem (ES) cells are a popular model system to study biological processes, though uncovering recessive phenotypes requires inactivating both alleles. Building upon resources from the International Knockout Mouse Consortium (IKMC), we developed a targeting vector for second allele inactivation in conditional-ready IKMC 'knockout-first' ES cell lines. We applied our technology to several epigenetic regulators, recovering bi-allelic targeted clones with a high efficiency of 60% and used Flp recombinase to restore expression in two null cell lines to demonstrate how our system confirms causality through mutant phenotype reversion. We designed our strategy to select against re-targeting the 'knockout-first' allele and identify essential genes in ES cells, including the histone methyltransferase Setdb1. For confirmation, we exploited the flexibility of our system, enabling tamoxifen inducible conditional gene ablation while controlling for genetic background and tamoxifen effects. Setdb1 ablated ES cells exhibit severe growth inhibition, which is not rescued by exogenous Nanog expression or culturing in naive pluripotency '2i' media, suggesting that the self-renewal defect is mediated through pluripotency network independent pathways. Our strategy to generate null mutant mouse ES cells is applicable to thousands of genes and repurposes existing IKMC Intermediate Vectors.

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