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Title: Assessment of nuclear reprogramming activity in mammalian ES cells
Author: Sullivan, Stephen
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2004
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A murine cell hybrid system was established and optimised to study nuclear reprogramming of somatic cells via fusion with embryonic stem cells. The system generated hybrid clones which displayed an ES phenotype and in which a somatically derived transgenic Oct-4 promoter had been reactivated. Methods were also developed to quantify heterokaryon formation, so that effects on the fusion process could be distinguished from effects on nuclear reprogramming per se. Hybrid cell lines displayed high endogenous alkaline phosphatase activity and expressed undifferentiated cell marker SSEA-1 but not markers associated with differentiated cells (SSEA-4 and CD90). These lines were pluripotent, demonstrating the ability to form the three embryonic lineages both in vivo and in vitro. This system was used to investigate whether several treatments (all either known or expected to perturb global gene expression patterns) affected nuclear reprogramming. Moderate heat shocking of thymocytes prior to fusion with murine ES cells resulted in increased hybridisation frequencies but, as fusion was also increased, it was impossible to verify whether an increase in nuclear reprogramming was partly responsible. Serum starvation of primary embryonic fibroblasts significantly increased nuclear reprogramming, as did ES cell confluence. Murine ES cells were found to lose their capacity to reprogram as they reached high passage numbers. Constitutive or transient expression of nucleoplasmin in murine ES cells did not increase their capacity to reprogram but instead led to increased cell death. Attempts were made to generate hybrids from human ES cells, but no hybrids were successfully generated. This was at least partly due to human ES cells being more difficult to fuse with other cells even using a range of different fusagens. Finally it was found that treating human ES cells with hyaluronidase prior to electropulsing resulting in five times more heterokaryon formation indicating that the extracellular matrix of these cells had prevented fusion. The methods developed here provide the basis for further study of the mechanisms underpinning reprogramming.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available