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Factors affecting the reprogramming efficiency of somatic cells
thesisposted on 08.02.2017, 03:39 by Tat, Pollyanna Agnes
Embryonic stem cells (ESCs) are pluripotent, that is, they are capable of indefinite self renewal in vitro while maintaining the ability to differentiate into cell types of all three germ layers. However, once the cells differentiate into somatic cells, pluripotency is lost. Reprogramming describes the reversion of a somatic cell’s differentiation status back to a state of pluripotency and has been demonstrated by the somatic cell nuclear transfer, cell fusion and induced pluripotency techniques. Despite recent advances, the reprogramming efficiency achieved by all methods remains extremely low. Therefore the main aim of this thesis was to investigate factors affecting reprogramming efficiency, including the in vitro age of cells, cell cycle synchronisation and somatic cell type. Firstly, the effect of different fusion methods on reprogramming efficiencies was investigated. Polyethylene glycol (PEG) and electro–fusions were conducted between murine somatic cells and ESCs. The effect of using late versus early passage somatic cells was also examined. Under the conditions defined, it was found that the reprogramming efficiencies were not significantly different between PEG and electrofusion, suggesting that the fusion method does not affect reprogramming potential. However, the reprogramming efficiency was significantly higher (15 fold greater, p>0.05) when using early passage compared with late passage somatic cells, indicating that senescence can affect the reprogramming potential of somatic cells. Next, it was hypothesised that synchronisation of the ESC and somatic cell cycles could improve reprogramming efficiency prior to fusion. ESCs were arrested at G2/M phase using the tubulin inhibitor, nocodazole and subsequently released from the drug to obtain relatively synchronous populations of ESCs at G2/M, G0/G1 and S phases (77.43%, 60.47% and 62.30%, respectively). Each of these synchronised ESC populations was fused to un-synchronised somatic cells. The results showed that the control group consisting of fusions between un-synchronised cells led to the highest reprogramming rate, implying that ESC cycle synchronisation may have limited utility in improving reprogramming efficiency. Chapter 5 investigated the effect of somatic cell type on fusion and reprogramming efficiencies. Different somatic cell types were tested including neural stem cells (NSCs), adipose tissue derived cells (ADCs), mesenchymal stem cells (MSCs) and mouse embryonic fibroblasts (mEFs). Following fusion, it was found that the reprogramming efficiencies differed greatly, with mEFs and MSCs showing highest efficiencies while NSCs and ADCs had significantly lower reprogramming efficiencies. Given these findings, chapter 6 aimed to determine whether the same somatic cells could be used to generate induced pluripotent stem (iPS) cells with the same or differing reprogramming efficiencies. Using retroviral transduction of the Yamanaka factors Oct4, Sox2, Klf4 and c-Myc to generate iPS cells, the reprogramming efficiency was found to be significantly higher for ADCs with an 8 and 38 fold greater efficiency than NSCs and mEFs, respectively. The conflicting results of relative efficiency between cell fusion and iPS reprogramming, highlights the different mechanisms of reprogramming of each technique. This thesis has shown that several factors can affect reprogramming efficiency, namely the choice of somatic cell type and age of cells. This facilitates the identification of different candidate somatic cell types for reprogramming. Moreover, this thesis has identified ADCs as a readily accessible source of cells for efficient iPS cell generation. This will be essential for feasible translational outcomes particularly when non-genetically modified approaches with even lower efficiency of iPS cell generation are adopted for the clinic.