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An mRNA-reprogramming method with improved kinetics and efficiency and the successful transdifferentiation of human fibroblasts using modified mRNA

Preskey, David Alexander (2017) An mRNA-reprogramming method with improved kinetics and efficiency and the successful transdifferentiation of human fibroblasts using modified mRNA. PhD thesis, University of Sheffield.

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Abstract

Induced pluripotent stem (iPS) cells have the potential to generate a wide array of cell types from multiple lineages that enable us to explore the mechanisms that are involved in the conversion of cell states. The reprogramming process that generates iPS cells is complex, but since its discovery, technical advancements and improvements in the methodology have improved the speed and efficiency of generating integration-free, clinically relevant iPS cells. However, despite improvements, the mechanisms of reprogramming are not fully understood and so the process remains largely inefficient and slow. It has been reported that reprogramming mediated through the delivery of exogenous mRNAs encoding OCT4, SOX2, KLF4 and cMYC is a fast and efficient method for generating integration-free iPS cells. Here we show that mRNA reprogramming can be enhanced further by employing an mRNA dose-ramping approach that provides greater control of the dose of mRNA that is introduced into the target cells. This improvement upon existing methods promotes the viability of the target cells during reprogramming which in turn improves the efficiency, speed and success of generating iPS cells. We also show that an optimisation to the reprogramming factor cocktail, replacing OCT4 with a fusion between OCT4 and the transcriptional activation domain of MYOD1 – called M3O, further improves the kinetics of reprogramming. Reprogramming disease cells is also possible in that several iPS cell-disease models have been established that have successfully modelled aspects of disease development in vitro. Here we show the applicability of using the mRNA approach we have developed, on neuroblastoma cells and the characterisation of iPS cells reprogrammed from neuroblastoma cells using OCT4, SOX2, KLF4 and cMYC delivered using Sendai viral vectors. Finally, we demonstrate how human fibroblasts introduced to a vector encoding MYOD1 causes them to transdifferentiate in to myoblast-like cells without a genomic footprint. Together this data demonstrates how integration-free mRNA can be used to control gene expression to direct cell fate through reprogramming and transdifferentiation. This mRNA approach provides proof of concept that warrants the testing of other genes to explore their function in reprogramming and other pathways that govern cell fate.

Item Type: Thesis (PhD)
Academic Units: The University of Sheffield > Faculty of Science (Sheffield) > Biomedical Science (Sheffield)
Identification Number/EthosID: uk.bl.ethos.723206
Depositing User: Mr David Alexander Preskey
Date Deposited: 29 Sep 2017 13:35
Last Modified: 12 Oct 2018 09:45
URI: http://etheses.whiterose.ac.uk/id/eprint/18242

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