TRANSCRIPTIONAL REGULATION OF THE DEVELOPING HUMAN PANCREAS

Abstract

Regenerative medicine is the restoration of normal physiological function in human cells, tissues or organs. Pluripotent stem cells can be cultured and differentiated to all somatic cells. Our work focused on the developing human pancreas. Specifically, by having a greater understanding of the in vivo process of development, we hope to recapitulate the process in vitro with the goal of generating mature pancreatic β-cells. Generation of mature β-cells will allow for the modelling and treatment of pancreatic disease, notably diabetes. To tackle this question, we asked, “What are the downstream targets of PDX1?” By analysing multiple human embryonic and differentiated progenitor derived hESC pancreatic datasets, we sought to answer this question. For the pancreas, analysis of human embryonic pancreatic tissue alongside existing differentiated samples has not been studied before. This vector allowed us to more directly probe the human system; in mouse there are differences in transcription factor expression profiles. Understanding of the human system will give us a more robust understanding for the generation of human beta cells going forward. I developed and implemented a bioinformatics pipeline that had the goal of sifting out interesting putative candidates of pancreatic differentiation centred around the progenitor stage. This included examining differential expression over time, in PDX1 WT and null pancreatic progenitors, human embryonic pancreas expression (CS17-19), as well as PDX1 and H3K27Ac binding information. We shortlisted a subset of genes and selected HES4 as a candidate gene. We generated CRISPR-Cas9 knock-out and knock-in mutants to probe expression and protein function. Our data tentatively reveal that transcription is sensitively regulated at the N-terminus of HES4. In addition, we reveal HES4 as an inhibitor acting as a gatekeeper for endocrine differentiation. HES4 null lines have increased expression of endocrine differentiation markers: PAX6, NEUROD1 and NKX2-2. The expression profiles for the endocrine markers suggest that different parts of the regulatory domains of HES4 interact with downstream transcription factors differently. The other genes output from the pipeline remain promising candidates for further studies in the search to understand the pancreas and treat diabetes.

Date of Award	1 Aug 2019
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Neil Hanley (Supervisor)