The role of adhesion molecule interactions in the engraftment of transplanted hepatocytes into host liver

In the UK liver disease is the fifth most common cause of death, and is rising dramatically. Whole organ liver transplantation is unfortunately limited in its availability. Other options include infusing suspensions of liver cells into patients, but the delivery of these cells within the liver needs to be improved as at present we can only deliver small numbers of cells.

I will identify the molecules which control the entry of infused liver cells into the liver, and increase their activity, thus improving the number of cells entering. Initially I will do this in a complex multi-cellular culture model which mimics what happens in a living organ. Having identified which molecules are most important in improving the engraftment of liver cells in tissue culture I will then proceed to a mouse model where I can test the effects of stimulating these molecules in a living organism. Of note I will use a mouse which has a metabolic liver disorder, which means that as well as being able to quantify the number of human liver cells which have engrafted within the mouse liver I can also see what impact they have had on the metabolic defect.

Chronic liver disease is an increasing cause of morbidity & mortality in the UK, for which liver transplant is increasingly insufficient due to the lack of donor organs. Experimental treatments such as hepatocyte cell transplantation are however limited by poor engraftment in the recipient liver. The entry of leukocytes into the liver is regulated by specific adhesion molecule/chemokine interactions with hepatic sinusoidal endothelial cells (HSEC), and I believe that similar mechanisms regulate the migration of hepatocytes across HSEC.

My hypothesis is that there are specific adhesive/migratory mechanisms which promote hepatocyte engraftment via the sinusoids and that they can be manipulated to improve the efficiency of hepatocyte transplantation.

I will screen for novel candidate molecules by microarray analysis of diseased liver tissue in order to identify specific ligands which may be important in hepatocyte engraftment/migration. Using flow cytometry I will examine primary human hepatocytes isolated from human liver tissue for expression of corresponding co-receptors to allow me to focus on potentially relevant co-expressed pairs only. Using this data I will then define the role of these receptors/molecules (such as 1-integrin, CXCR3, CD44) in established flow-based adhesion and trans-migration assays which model hepatocyte transplantation in vitro. I will manipulate these receptor interactions to improve the efficiency of cell adhesion/trans-migration.

I will apply these manipulations to human hepatocytes prior to transplantation into the Fah-/-/Rag2-/-/Il2r -/- mouse which develops tyrosinaemia, and thus provides a selection advantage for proliferation of engrafted human hepatocytes. It is also T-/B- and NK-cell deficient thus preventing a xenorejection response against human hepatocytes. I will initially use intravital microscopy (IVM) to study interactions between intravenously administered hepatocytes and sinusoids in real time in vivo to define the mechanisms regulating engraftment. Finally, in order to define the impact of receptor manipulations on longer-term engraftment, FRG mice will receive 1?106 human hepatocytes via intraportal infusion.

At completion of this project I will have addressed one of the major hurdles to the use of hepatocyte transplantation, and be well positioned to consider preliminary clinical studies.