The Transcriptional Profile of Microglia: from Brain to Dish Fiona Elizabeth Calvert

Microglia are the tissue resident macrophages of the central nervous system (CNS) and multiple lines of evidence indicate that microglia are a pathogenic cell type in Alzheimer’s disease (AD). It is important to understand the transcriptional profiles of microglia, both from primary human cells and the in-vitro model systems used to study the cells at scale. In this thesis, I aim to build on previous small-scale studies of primary microglia and in-vitro model systems to answer three major questions: 1. Can transcriptional data from fresh, primary human microglia be used to identify novel subpopulations of cells and understand how clinical phenotypes influence gene expression? 2. How accurately do current simple in-vitro model systems of human microglia capture the profile of primary human cells? 3. Do more complex model systems move cultured cells further along a trajectory towards the primary cell type?

I have utilised RNA-sequencing technology to build the most comprehensive transcriptional profile of primary human microglia to date, from over 100 neurosurgical patients. Using single-cell sequencing I have demonstrated that clinical pathology, particularly major trauma, causes specific gene expression changes within microglial transcriptomes. I have then shown that in-vitro models of primary microglia have significantly reduced expression of key marker genes and transcription factors, such as P2RY12 and SALL1, when compared to primary cells. Using gene-set enrichment analysis tools, I have shown that many of the genes with higher expression in primary cells can be linked to neuronal processes such as CNS myelination. Data from the third chapter of this thesis identified the CNS environment as a major stimulating factor in the gene expression profile of primary microglia. Therefore, I used single cell analysis to understand how culturing stem cell derived microglia in the presence of neurons could move in-vitro systems closer towards the primary cell type. In summary, the work in this thesis has demonstrated that microglial transcriptomes are constantly reacting to stimuli within the local CNS environment, both to maintain their unique gene expression profiles and to respond to clinical conditions. I have also shown that current in-vitro model systems do not fully capture this transcriptional profile which largely appears to be driven by environmental stimuli within the CNS.