Investigating the role of the Polycomb Repressive Complex 2 in human in vitro pancreatic differentiation

The potential to use human pluripotent stem cells in regenerative medicine is an idea that continues to excite and captivate much of the science community, decades after the initial discovery of human embryonic stem cells. This interest has grown with the discovery of induced human pluripotent stem cells, combined with the continual improvement of differentiation protocols. These protocols allow the formation of an ever-increasing pool of mature and functional cell types within an in vitro laboratory environment. However, a number of important questions still exist in terms of the production of these cells, including their functionality and how closely these cells resemble the in vivo counterparts. One such area still to be explored is the epigenetic aspect of the in vitro derived cells. Beyond this understanding, the potential to apply epigenetic modulation to improve differentiation programmes, and/or the final cell product, is an important area of research. In this dissertation, I use the in vitro production of human pancreatic endocrine cells as a model to study histone methylation changes, and associated transcription, of differentiating cells produced in culture from a pluripotent stem cell population. I describe the dynamic epigenetic and transcriptional changes that occur in a bulk population of human pluripotent cells developing through a number of intermediary cell types to form pancreatic endocrine cells. This analysis was then used to study the differences between in vitro end-cell populations and in vivo derived cells, highlighting the large variation in gene expression and persistent H3K27me3 modifications present in the in vitro cells. I show that manipulation of Polycomb protein-mediated H3K27me3 levels in differentiation at specific time-points has a variable effect on end-cell population, with an apparently negative effect early in differentiation, but with potentially a more positive effect later in the differentiation. Lastly, I was able to introduce a reversible knockdown system within the human pluripotent cells, which will allow us to study the functional role of Polycomb-proteins in the context of in vitro differentiation. In summary, my work has established a role of aberrant histone modifications in limiting in vitro differentiation capabilities and provides a new framework for manipulating epigenetic processes to improve differentiation outcomes that may be relevant for improving targeted cell production