Understanding the determinants of fate potential in human cardiac progenitor cells.

Abstract

Naomi R Phillips. Doctor of Philosophy. Final submission June 2022. Understanding the determinants of fate potential in human cardiac progenitor cells. Heart disease is the leading cause of death worldwide, in part due to the adult heart’s lack of regenerative capacity. During development, cardiac progenitor cells (CPCs) generate the range of cell types required for proper cardiac function. However, CPC populations are heterogeneous and currently lack molecular definition. To efficiently generate defined multipotent CPCs from hPSCs we need to better understand their development and fate determinants. Cardiac differentiation was achieved using the NKX2-5eGFPSOX17dTomato hESC line, with upregulation of cardiac genes and corresponding chromatin accessibility in NKX2-5+SOX17- populations at day 8. I showed that modulation of Activin and BMP signalling during early lineage specification can influence anterior-posterior patterning from a primitive streak-like stage, generating progenitors with a range of fate potentials. A small population of NKX2-5+SOX17+ cells was determined to be endoderm-derived NKX2-5+ thyroid lineage cells. Using a MEIS1/2 KO hESC model I have shown that MEIS transcription factors are crucial for cardiac differentiation due to their role in increasing chromatin accessibility of GATA and SMAD mediated regulatory regions. MEIS1/2 KO significantly reduced NKX2-5 expression due to a lack of chromatin accessibility around the locus, highlighting the importance of chromatin states for gene regulation. To understand cardiac NKX2-5 regulation more specifically, I interrogated ATAC-seq and ChIP-seq datasets and uncovered eight potential enhancer regions of NKX2-5. Luciferase assays suggested that six of these enhancers had activity in human cardiomyocytes, with a range of mechanisms for tissue specific activity. Finally, I explored self-assembling peptide hydrogels as a 3D CPC culture substrate, making a step towards therapeutic and disease modelling applications. To deepen the understanding of CPC fate determination, further examination of the link between pre-NKX2-5 molecular identity and fate potential in hPSC derived CPCs is required. In addition, further time-dependent analysis of putative enhancer regions is required. Although not conclusive in nature, in this project I have made significant progress towards these goals.

Date of Award	1 Aug 2022
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Alberto Saiani (Supervisor) & Neil Hanley (Supervisor)