Regulated mis-expression of PDX1 and NKX6.1 during pancreatic differentiation of pluripotent stem cells

2017-03-01T05:38:36Z (GMT) by Tan , Gemma
Human pluripotent stem cells represent a potential source of pancreatic beta cells for the treatment of type 1 diabetes. Methods for generating pancreatic endoderm from pluripotent stem cells are becoming increasingly robust, although the general applicability of any given method to a broad range of stem cell lines remains a challenge. Therefore, a deeper understanding of the molecular mechanisms controlling the commitment of cells to pancreatic endoderm is desirable. A key step in this process is the co-ordinated up-regulation of two transcription factors, PDX1 and NKX6.1; factors that together identify cells fated to become pancreatic endoderm and to later give rise to endocrine cells. In this thesis, we examined the effects of overexpressing PDX1 and NKX6.1 during the course of PSC differentiation. We employed the use of a number of transgenic systems, namely the doxycycline inducible Tet-On expression system, Destabilisation Domains (DD) and Estrogen Receptor (ER) fusion proteins. Our experiments demonstrated a potential for each of the 3 expression systems, however we encountered drawbacks within each one preventing us from establishing a definitive conclusion as to the affects of over-expressing PDX1 and NKX6.1 during PSC differentiation. We conclude that only once both the expression systems and differentiation protocols are optimised will it be possible to determine the effects of co-expressing these two factors during the course of pancreatic differentiation.