Investigating liver regeneration using single cell RNA sequencing

Liver disease causes over 2 million deaths world-wide each year and is the most common disease related deaths in adults aged between 35-49 years old in the UK. While the liver has a large capacity to regenerate this is overwhelmed during chronic injury. Currently there is no drug-based treatment for end stage liver disease, with transplantation the only option. This is problematic as only around 10% of the global transplantation needs are met and urgent new therapeutics are required to tackling this highly prevalent disease. Despite decades of research elucidating the mechanisms behind the liver’s natural capacity to regenerate, there has not yet been a therapeutic agent approved. The prominent feature of liver regeneration is the replication of mature hepatocytes. Research has assumed homogeneity in function among the hepatocyte population during regeneration, however recent studies have shown that there is a more heterogeneous response. More work is required to understand the complex nature of this process at a higher resolution to unpick any heterogeneity and discover any further regenerative signals. Therefore, this project aimed to use a single cell RNA sequencing approach to identify any potentially novel heterogeneous populations of hepatocytes during liver regeneration and further identify any key signalling molecules. Replicating and non-replicating hepatocytes were sorted from a partial hepatectomies FUCCI2a mouse model which reports G1 cells with a mCherry signal and S/G2/M cells with a mVenus signal. Hepatocytes were sorted directly into 384 well plates for single cell sequencing. Unsupervised cluster was performed to identify several populations of hepatocytes. Non-replicating cells split into cluster denoting there spacial location in the liver lobule. This was identified through the gradient of expression of zonal marker genes. While no novel heterogeneous cluster of replicating hepatocytes were found, several novel signalling molecules such as Il33, Il15, Dll1, Cklf, and Bmp7, were identified to be expressed only by replicating hepatocytes. Furthermore, a population of “primed” mCherry positive hepatocytes appeared to express ii several cell cycle marker genes. This population could represent a population of cells captured just before entry into S phase or a priming action of hepatocytes to prepare for cell cycle entry, awaiting an appropriate initiating signal. Interesting this population showed high expression for a circadian rhythm associated gene Timeless. Replication during partial hepatectomy is believed to be highly synchronous and Timeless may be the protein that controls the timing of replication during the model. Further studies of these genes are required to understand their function and importance in liver regeneration. In conclusion, the work demonstrated within this thesis highlights the need to study liver regeneration using high resolution techniques such as single cell RNA sequencing