Stable or transient transfer?

To investigate longevity of both substrates and ascertain whether transfer is stable or transient, hEKs were maintained in their original wells for 10 days following nucleofection. A similar pattern emerged between the two

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substrates with expression peaking for both constructs after 3 days and decreasing over time. When hEKs received miRNA inhibitor, this molecule remained in approximately 80% hEKs after 10 days, while GFP-P had largely been cleared by this point. This may be due to the initially increased cytoplasmic concentration of miRNA inhibitor retaining detectable intensity after several round of mitosis.

To confirm transfer was transient and also assess the feasibility of successive rounds of nucleofection, hEKs were subject to a further round of nucleofection. hEKs that received either GFP-P or miRNA inhibitor demonstrated an increased number of successfully nucleofected cells and in the case of miRNA inhibitor, an increase in fluorescence was also observed. This suggests an increase in substrate transferred to each cell and that hEKs nucleofected during initial nucleofection were more amenable to receiving substrates during serial nucleofections. In addition, hEKs nucleofected in the absence of either GFP-P or miRNA inhibitor during the second round demonstrated an absence of either substrate, providing confirmation that transfer is transient and substrates are eventually cleared through mitosis if not repeatedly transfected [205].

Co-nucleofection has been adopted by many groups as an effective way of delivering a number of substrates [206-208] and it was hypothesised that to develop an effective reprogramming strategy it may be necessary to deliver a number of inducing stimuli. During this investigation it was possible to demonstrate successful delivery of both GFP-P and miRNA inhibitor to hEKs. Flow cytometry revealed that 7.64% of adherent hEKs received both constructs. Considering the low levels of nucleofection reported in other strategies, with only 10.8% of hASCs receiving the minicircle vector before undergoing reprogramming [144] it is not unreasonable to consider co- nucleofection of plasmid DNA and miRNA inhibitors as a practicable strategy for reprogramming hEKs.

172 4.4 Inhibition of miR-145

miR-145 was highlighted early in this study as a potential mediator of pluripotency [119]. It was hypothesised that modulating its expression may alter the expression of key pluripotency regulators. This investigation successfully demonstrated that inhibition of miR-145 activated pluripotency genes in hEKs that are normally silenced. In the case of Oct4, expression was significantly upregulated following miR-145 inhibition, with an expression peak after 5 days. Previous studies have focused on initiating reprogramming through introduction of a number of genes with the vast majority including Oct4 [45, 144]. Another study, which mirrors the level of transcriptional regulation described herein, utilised small interfering RNA (siRNA) to mediate expression of scaffold attachment factor A (SAF-A) [209]. This study demonstrates that decreasing SAF-A mRNA through siRNA transfection subsequently decreased Oct4 expression and vice versa This supports the findings presented in this thesis, which suggest that indirect activation of Oct4 expression can replace exogenous Oct4. The majority of reprogramming strategies involve the induction of Oct4, consistent with its role as a mediator of a pluripotent state suggesting that Oct4 is involved in transcriptional activation of itself or as a heterodimer with Sox2.

Literature has shown that miR-145 is associated with both Oct4 and Sox2 [119] therefore discovery that miR-145 is capable of activating Oct4 expression in hEKs suggested that miR-145 may mediate expression of other pluripotency associated genes. As hypothesised, expression of Sox2 was found to have significantly increased upon miR-145 inhibition. The pattern of expression was different to that observed for Oct4 with a progressive increase peaking at 9 days, with more than a 25 fold increase detected compared to hEKs nucleofected in the absence of miR-145 inhibitor. If we consider another direct target of miR-145, Klf4, we

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observed a similar pattern of re-expression to that of Sox2. Expression gradually increased over time and peaked at more than 25 fold after 9 days, which is distinctly similar to Sox2 re-expression. With previous studies highlighting miR-145 as directly targeting Oct4, Sox2 and Klf4 3‟ UTRs [119] it is not surprising that miR-145 inhibition produced this biologically significant effect. Whilst miR-145 directly targets all three genes, the fact that Oct4 does not follow the same pattern of re-expression nor reaches the same level is not surprising given the tight multifactorial regulation that this gene demonstrated in previous studies [62, 210].

The interaction between miR-145 and c-Myc has not received as much attention as its effect on Oct4, Sox2 and Klf4. However, miR-145 has been shown to directly silence c-Myc, an oncogenic phosphoprotein, which plays a key role in regulating cell cycle progression, apoptosis and transformation [211]. Inhibition of miR-145 resulted in a significant increase in c-Myc expression, which peaked after 5 days and exceeded more than 10 fold upregulation. This echoes another study whereby miR-145 inhibition enhanced c-Myc expression. This study demonstrated the link between p53 and c-Myc by identifying that p53 transcriptionally induces expression of miR-145 through interaction with a p53 response element in the miR-145 promoter. miR-145 then proceeds to silence c-Myc, however this process is reversible demonstrated by miR-145 inhibition upregulating c-Myc expression via p53 [212].

There have been no reports of miR-145 directly targeting Nanog, however it was hypothesised that Nanog expression may be significantly altered as a result of increased levels of Oct4 and Sox2 given the intrinsic, cooperative regulation of these master regulators [181]. Gene expression data did not conclusively indicate Nanog re-expression, despite a statistically significant upregulation in Nanog expression detected after 1 and 7 days. This suggests that miR-145 inhibition alone is not sufficient to induce pluripotency.

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To ascertain whether miR-145 inhibition had altered the genotype of hEKs, the expression of the ubiquitously expressed cytokeratin-14, was investigated. qRT-PCR analysis revealed an initial decrease in expression, which increased over time resuming to basal levels after 9 days. This reflects the transient nature of miRNA inhibition and provides further evidence that miR-145 inhibition is insufficient to induce a pluripotent state.

4.5 hESC media formulation promotes pluripotency gene expression