The ability to reprogram somatic cells toward a stem cell phenotype has created a powerful method of reversing the fate of lineage restricted cells. If the potential of reprogramming is realised we will be able to provide a source of autologous, functional cells circumventing many of the issues associated with donor transplantation and provide a source of stem cells free from the controversy surrounding derivation from embryos. MicroRNA (miRNAs) are an attractive substitute to foreign DNA for induction of reprogramming. This thesis focused on a specific miRNA, miR-145 due to its role as a repressor of pluripotency during embryogenesis. The majority of reprogramming approaches to date have utilised viral vectors to deliver inducing stimuli however a considerable amount of literature strongly suggests that cells reprogrammed using viral vectors are unsuitable from a clinical perspective due to risks associated with insertional mutagenesis and potential immunogenicity. Therefore this investigation sought a non-viral method of delivering inducing stimuli and an electroporation based technique, known as nucleofection was identified. This non-viral delivery method was evaluated in terms of its efficiency and cell viability and was found to be highly effective in its capacity to deliver both plasmid DNA and miRNA inhibitors to human epidermal keratinocytes (hEKs). Nucleofection of hEKs with an antisense oligonucleotide, which effectively inhibits miR-145 was shown to significantly increase the expression of Oct4, Sox2, Klf4 and c-Myc all of which are required for the successful reprogramming of somatic cells. MiR-145 inhibited hEKs were subsequently subjected to modified culture conditions in an attempt to further promote the expression of pluripotency-associated transcription factors. hESC media containing growth factors, known to promote a human embryonic stem cell phenotype, lead to a distinctive change in cell morphology with cells clustering together to form colonies and also further stimulated the expression of pluripotency associated genes. While hEKs did exhibited re-expression of normally silenced pluripotency genes they had not undergone full cellular reprogramming however now presented a more ‘plastic’ phenotype. A direct reprogramming approach was formulated where miR-145 inhibition of hEKs was performed and post-nucleofection cells were incubated using either standard hEK media or growth factor supplemented hESC media for 5 days. Defined osteogenic, chondrogenic or adipogenic differentiation media were then applied to stimulate reprogramming towards ‘tri-lineage’ fates, whilst crucially demonstrating transgermal differentiation of hEKs. A defined neurogenic media was also applied to further validate their differentiation potential and together this provided further evidence for acquisition of a ‘plastic’ phenotype with identification of lineage specific markers following directed differentiation. Taken together this investigation details a novel, miRNA mediated strategy for the direct reprogramming of hEKs in the absence of foreign DNA or viral vectors whilst facilitating re-differentiation into a number of desirable lineages offering significant advances for clinical translation.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:564228 |
| Date | January 2012 |
| Creators | Lewis, Fiona |
| Contributors | Hunt, John; Rhodes, Nick |
| Publisher | University of Liverpool |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://livrepository.liverpool.ac.uk/7073/ |
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