A single-cell sequencing approach to identify new therapeutic targets to drive liver regeneration

Liver disease is a major cause of morbidity and mortality and liver transplant is still the only treatment option for end-stage liver disease. With limited numbers of donors and the high cost of transplantation, it is essential an effective pro-regenerative therapy is developed.
The liver is unlike most human organs in that it has a remarkable regenerative capacity, largely mediated through hepatocyte replication. The liver is comprised of 80% hepatocytes and 20% non-parenchymal cells which include kupffer cells, endothelial cells and stellate cells. Hepatocytes are also unusual in that they exhibit varying levels of ploidy and nuclear number. Rather than being a solely 2N (diploid) population, a hepatocyte populations display a range of ploidies from 4N up to 16N, however the majority are 4N. Alongside this, hepatocytes can be mono or bi nuclear. During regeneration in mice following 2/3 partial hepatectomy it has been shown that 95% of hepatocytes enter the cell cycle, however not all complete cell division. It is therefore unclear why some cells complete cell division, helping regenerate the liver, and others do not. This project will therefore aim to expand our knowledge of hepatocyte biology during liver regeneration using single cell transcriptome sequencing. This will allow investigation into whether hepatocytes display functional heterogeneity, and should facilitate the identification of pro-regenerative therapeutic targets that can be used to drive liver regeneration.
The hypothesis is that hepatocytes display significant functional heterogeneity in healthy and diseased livers, and this functional heterogeneity is regulated by hepatocytes ploidy and nuclear number.

Therefore I will aim to answer these three questions:
1. Do hepatocytes display functional heterogeneity?
2. Does ploidy level regulate functional heterogeneity?
3. Does nuclear number regulate the transcriptome in hepatocytes with the same ploidy level?

Hepatocytes can be easily separated by ploidy using a FACS cell sorter and Hoechst DNA staining. The greater the Hoechst staining observed the higher the level of ploidy. Single hepatocytes will be isolated from both healthy and injured mouse and human livers and sorted into individual wells of a 384 well plate and sent for transcriptome sequencing. This will allow a deep level of analysis of how ploidy affects hepatocyte biology in both homeostasis and injured/regenerating livers. The mouse injury model I will use is the 2/3 partial hepatectomy model of liver regeneration. This is a standard model in the field. Human liver samples will be collected from explant livers during liver transplantation. Sequencing data will be analysed using in-depth informatics and statistical approaches.
Unfortunately, isolating cells base on nuclearity is more complex. Currently there is no high throughput method of separating mono- and bi-nuclear cells. Cells can be individually isolated using micro pipetting, however if a high throughput method could be identified, this would be more desirable. During this project I will investigate this area and attempt to set up a high throughput approach. The population of most interest for this area of study is the 4N population. Study of the 4N population will allow direct comparison of mono- and bi-nuclear 4N hepatocytes. By concentrating on these populations initially, ploidy level is constant between the two populations. Cells will be first sorted based on their ploidy status, 4N cells will then be analysed under a microscope for nuclear number, and individually picked using a micro pipetting technique or sorted using a high throughput technique once this is developed. These mono- and bi-nuclear 4N hepatocytes will then be sequenced and analysed using the same informatics and statistical approaches as listed above.