One hallmark of embryonic stem cells (ES) is their ability to renew themselves indefinitely and still retain the potential to develop into any specialized cell type once triggered by specific exogenous signals. This very versatile nature has made them an attractive model to study developmental events occurring during embryogenesis and also to employ them for regenerative medicine. The idea to exploit their developmental potential for intended therapeutic applications requires a detailed knowledge of the molecular regulation of differentiation. Thus pluripotent embryonic stem cells can be employed to investigate the molecular framework regulating pluripotency. The major aim of this research endeavour is to explore the role of DNA methylation in regulation of endogenous Sox2 transcription factor in context of mouse ES cells following their transition from the pluripotent to a differentiated state. An insight into molecular regulatory mechanisms will shed light on developmental programming and also aid in refining of methodologies for differentiation and nuclear reprogramming increasing their chances of success and efficiency. Mouse embryonic stem cells were differentiated towards osteogenic and neural cell types through the formation of embryoid bodies (EBs) – cellular aggregates partially recapitulating the early embryonic development. These EBs were then disaggregated and single cells plated in medium containing supplements to promote osteogenic or neural differentiation while control cells were grown in medium lacking those factors. Cells were harvested undifferentiated and at different time points during differentiation. Molecular characterization was carried out by expression profiling of lineage specific genes and proteins using RT-PCR and immunofluorescence respectively. DNA methylation analysis of two regulatory regions of Sox2 i.e. SRR1 and SRR2 was carried out by MS-PCR and bisulphite sequencing. Embryonic stem cells were observed to be differentiating as evidenced by changes in cellular morphologies and lineage-specific markers expression. Two regulatory regions of Sox2, namely SRR1 and SRR2, were found to be methylated by methylation sensitive PCR at all time-points chosen for analysis in differentiating cells. Three individual CpGs in SRR2 region were then analysed further by bisulphite sequencing which appeared unmethylated in both undifferentiated and differentiated embryonic stem cells. This hints towards the possible role of DNA methylation in regulating the expression of Sox2 in differentiating embryonic stem cells and need further investigation.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:588365 |
| Date | January 2013 |
| Creators | Batool, Sajida |
| Publisher | University of Nottingham |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://eprints.nottingham.ac.uk/13551/ |
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