The epicardium is a transcriptionally heterogeneous cell layer covering the heart, crucial to correct cardiovascular development. Following epithelial-to-mesenchymal transition (EMT), epicardial cells migrate into myocardium, form coronary smooth muscle cells and cardiac fibroblasts, and instruct cardiomyocytes to proliferate and mature. Adult mammalian epicardium is quiescent, but reactivates post-injury with limited effect. However, in zebrafish and in neonatal mouse, epicardial signalling enables robust cardiac regeneration after myocardial infarction. We hypothesise that manipulating human epicardial function could facilitate heart regeneration, via reactivation of embryonic processes. However, epicardial regulation remains incompletely understood; although understanding epicardial mechanisms could be key to potentially manipulating epicardium for therapeutic benefit. This PhD investigates a candidate transcription factor, Basonuclin 1 (BNC1), in functional regulation of human epicardial models, and identifies this gene as a potential key human epicardial regulator. Epicardial-like cells derived from human pluripotent stem cells (hPSC-epi) were previously used for single-cell RNA sequencing (scRNA-seq) in order to investigate possible human epicardial heterogeneity. This identified two distinct hPSC-epi subpopulations: one high in WT1 expression, the other high in TCF21. Bioinformatic analyses identified BNC1 as a potential key node in the hPSC-epi signalling network, via network inference modelling. BNC1 is a transcription factor known to regulate migration and proliferation in other epithelia. Given our network inference analyses and the literature evidence, I hypothesised that BNC1 would have functional relevance in human epicardium, so aimed to investigate its function in hPSC-epi differentiation and epicardial cell migration, as well as identify its putative epicardial targets. Firstly, scRNA-seq data describing hPSC-epi heterogeneity were validated in primary human foetal epicardium and BNC1 expression was confirmed in human epicardial models. BNC1 was subsequently investigated, both by siRNA-knockdown in hPSC-epi and foetal epicardial explants and inducible knockdown cell lines (siKD). siKD hPSC-epi had over 90% BNC1 reduction and displayed significantly altered expression of canonical epicardial genes WT1 and TCF21: hPSC-epi heterogeneity was thereby lost. Altered hPSC-epi proliferation and viability were also observed. siKD hPSC-epi was subsequently used in a simple epicardial EMT model (epi-EMT). siKD epi-EMT displayed impaired migration and pronounced cortical actin localisation. ChIP sequencing and bulk RNA sequencing identified potentially promising BNC1 targets, such as actin-binding protein supervillin, for future investigation. We conclude that BNC1 is a key functional epicardial regulator in vitro, paving the way for in vivo characterisation. The knowledge that manipulating BNC1 regulates epicardial heterogeneity and function may instruct efforts to harness epicardial potential for future therapeutic benefit.The British Heart Foundation fully funded this doctorat
