Developing lentiviral tools to generate and characterise induced pluripotent stem cells

• Main Author: Kelly, Maebh
• Published: University of Bristol 2012
• Subjects: 616.02774
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.601177

Induced pluripotent stem cells can be generated by the forced expression of certain pluripotency factors, but little is known about the molecular changes underpinning this process. Micro-RNAs regulate many cell processes and have recently been show to have a role in the generation of iPS cells. In this thesis, we firstly set up a protocol to generate iPS cells in our lab and then investigate a role for stem cell specific miR-371 in the generation of pluripotent stem cells. Overexpression of miR-371 in addition to OCT4, SOX2, NANOG and LIN28 promotes pluripotency. MiR-371 directly targets RBL2 causing an increase in DNMT3b levels and also has anti-apoptotic effects, which may go some way towards explaining the effect of miR-371 on the pluripotency process. Furthermore, it was also noted that miR•371 also regulated expression of a novel intronic miRNA miR-1301, which is co-expressed with its host DMNT3al and highly expressed in neural stem cells. MiR•1301 in turn targets MECP2, reducing its expression and promotes neurite outgrowth from DRG cultures. However inhibition or overexpression of miR•1301 in neural stem cells does not affect the number of neurons or their length. These findings indicate that miR•371 has an important role in the generation and maintenance of stem cells and for the first time characterised and observed a functional effect of miR• 1301. The role of miRNAs in controlling cells fate is a complex one and this thesis goes some way towards unravelling some of the connections and will help with our overall understanding of the molecular mechanism behind changing cell states.