Baker, Andrew

Ponting, Chris

McCracken, Ian Richard

2022-06-15T09:07:52Z

2022-06-15T09:07:52Z

2022-06-14

The endothelium comprises the luminal surface of all blood and lymphatic vessels and is known to first emerge from mesodermal precursors in a process known as vasculogenesis. Following formation of a primitive vascular plexus, the endothelium rapidly expands by angiogenesis and undergoes specification into arterial, venous, capillary, lymphatic, and haemogenic subtypes. Additionally, in response to intrinsic and tissue specific cues, the endothelium undergoes further specialisation to meet the individual requirements of the surrounding tissue. However, despite improving understanding of the sequence of events occurring during endothelial cell (EC) development, the transcriptional control underlying these processes remains largely uncharacterised. The differentiation of human embryonic stem cells (hESC) to endothelium offers an appropriate model to study the transcriptional control of endothelial differentiation in vitro. In this study I applied high throughput single cell RNA sequencing (scRNA-seq) to an efficient 8-day hESC endothelial differentiation protocol, collecting cells at days 0, 4, 6, and 8. Flow cytometric analysis of day 8 cells revealed a population in which 66% of cells co-expressed endothelial markers CD31 and CD144. Analysis of scRNA-seq data from day 0 and 4 revealed homogeneous populations defined by expression of pluripotent and lateral mesoderm markers, respectively. In contrast, scRNA-seq analysis of cells collected at days 6 and 8 show the emergence of distinct endothelial and mesenchymal populations. Repeating scRNA-seq analysis of hESC-endothelial differentiation with a second hESC line as well as using an alternative differentiation protocol revealed highly comparable transcriptional signatures of endothelial differentiation. However, comparison of data from hESC derived endothelial cells (hESC-EC) to equivalent scRNA-seq data from cultured and freshly isolated human organ-specific endothelial cells, demonstrated a clear distinction in their transcriptional state, thus highlighting the likely unspecified nature of hESC-EC. Following on from these findings, I then sought to determine if mapping the dynamic transcriptional landscape of the endothelium during human cardiac development could be used to identify novel regulators of endothelial maturation and specification. CD31+/CD45- endothelial cells isolated by FACS from heart tissue obtained from 13- and 14-week fetuses were processed for scRNA-seq analysis. Subsequent dimensionality reduction and clustering of data revealed distinct endocardial, capillary, venous, arterial, and lymphatic populations each with a unique transcriptional signature. Application of trajectory inference methods predicted a minor endocardial contribution to the coronary vasculature via a venous population, prior to the subsequent arterial specification of capillary EC. Mapping differentially expressed genes over pseudotime revealed the expression of MECOM to coincide with the onset of arterial specification. In conjunction, gene regulatory network analysis also placed MECOM amongst known regulators of arterial EC specification such as HEY1 and SOX17. Subsequent knockdown of MECOM in arterial-like hESC- EC suggested a function in repressing venous identity in arterial EC. Together these studies provide a comprehensive map of the transcriptional landscape accompanying both hESC-EC differentiation and during human cardiac EC development. Mapping these dynamic processes offers new insight into the mechanisms underlying endothelial development, creating future opportunities for vascular regeneration for ischaemic disease. Lastly, in response to the COVID-19 pandemic I determined the susceptibility of endothelial cells to in-vitro infection with human coronaviruses, including SARS-CoV-2. Despite demonstrating productive infection of endothelial cells by HCoV-229E, a lack of replicative infection was observed by SARS-CoV-2. This was most likely due to an absence of the expression of the SARS-CoV-2 receptor, angiotensin converting enzyme 2 (ACE2).

https://hdl.handle.net/1842/39105

http://dx.doi.org/10.7488/era/2356

en

The University of Edinburgh

Mapping the transcriptional dynamics of the endothelium during human embryonic stem cell differentiation and cardiac development using single cell RNA sequencing

Thesis or Dissertation

Doctoral

PhD Doctor of Philosophy