LEANNE HODSON – ASSOCIATE PROFESSOR
Oxford Centre of Diabetes, Endocrinology and Metabolism (OCDEM)
University of Oxford
MEET THE TEAM
PROJECT TITLE: Development and validation of a human hepatocyte model to investigate how liver fat metabolism influences metabolic disease
PROJECT TIMESCALE: 3 years – 1 March 2013 to 28 February 2015
POST DOCTORAL FELLOW: Dr Charlotte Green
After two years of working on this project, which was generously funded by the Trust, we would like to take this opportunity to let you know what we achieved. We set out with the ambition of using human liver cells (hepatocytes) to develop a model that would allow us study the mechanisms that may play a role in causing accumulation of fat within liver cells. We were interested in undertaking this work as liver fat accumulation (hepatic steatosis) is associated with excess fat (triglyceride) in the blood, and this is a risk factor for heart disease (CVD) and diabetes. I am very pleased to say that we have been both productive and successful during this time and have formed strong collaborative links with surgeons, pathologists and other research scientists.
Underpinning the success of the project was the possibility to employ Dr Charlotte Green as a postdoctoral fellow. Although Charlotte possessed an excellent experience inin vitro cell culture techniques she had not previously isolated or worked with human hepatocytes. These cells are renowned for being tricky to work with and I was fortunate that Charlotte was not fazed when challenged with the task of setting-up, from scratch,the isolation and culturing methodologies. It can take years of experience to develop new techniques, particularly when the cells you are working with are sensitive to being isolated from liver tissue and then are very fussy about the conditions they are cultured in. Charlotte not only developed but adapted the isolation methodology so we can use small (>15g) pieces of ‘resection surplus tissue’ (that is human liver tissue from surgery that would be thrown away) to get a good yield and viability (~75%) of cells meaning even small pieces of tissue are useful for studies. We have demonstrated that even when taken out of the human body, isolated liver cells maintain specific metabolic traits. This finding is important as it allows us to study the effects of specific nutrients, such as fats and sugars, on fat metabolism pathways at the cellular level.Earlier this year we had this work accepted for publication in the American Journal of Physiology (Endocrinology and Metabolism). We have also presented the work at conferences where it has been very well received; Charlotte was awarded one of the ‘top’ abstract prizes for her talk at the British Association for the Study of the Liver (BASL) Basic Science Retreat in Devon last year.
The human liver cells models developed over the course of our project are now routinely used as they elegantly complement the human in vivo studies we are undertaking. This work has also provided an important platform for development of additional specialist skills for staff and students.
For example, Catriona McNeil, a research assistant within my group, who had no previous experience working with human cells, is now very competent in these methodologies. Finally, we would like to take this opportunity to thank the Trust and Trustees again for their support, it has been very much appreciated and provided the opportunity to gain invaluable pilot data for future grant applications, along with providing new collaborative opportunities.
INTERIM UPDATE – April 2014
Over the course of the last year we have made, what we believe, to be some very exciting progress on the project. We have moved closer to establishing a human hepatocyte model that will aid in understanding the processes involved in liver fat accumulation. Liver fat accumulation (steatosis) is a risk factor for cardiovascular disease and diabetes but the reasons why some individuals accumulate fat in their liver are poorly understood. Our method utilises excess (healthy) liver tissue (resection surplus) from consented patients undergoing liver surgery that would normally be discarded. The most established method for isolation of hepatocytes requires a large amount of tissue (100g or more) which has proved difficult to obtain; this has required we take a new approach. Over the past year Dr Charlotte Green, has worked very creatively and established methodology in which human hepatocytes can be isolated from small amounts (more than 15g) of liver tissue, to produce a relatively constant supply of human hepatocytes with little waste. The next obstacle encountered was removal of the red blood cells (RBCs) from the liver tissue. RBCs can be toxic and cause cell death to hepatocytes in culture.
Very recently Dr Green has solved this problem and we now have a robust method that provides a pure and healthy population of hepatocytes to work with; this has been a major step forward. How can we ensure we are getting healthy hepatocytes in the preparations? There are a number of ways to assess the viability (health) and functionality of cells, we check if specific ‘components’ are still working. For example we are interested in the mitochondria (energy generation factory) of the cells and by using a fluorescent dye (called TMRM) that is taken up into cells and stains only the healthy mitochondria (Figure 1), we can check functionality. As hepatocytes are fussy about the conditions in which they are cultured we are now moving on to investigate different culturing conditions (e.g. the amount of fat and/or glucose in the media) in order to optimise the conditions that best recapitulates their environment inside the human body. We hope this will provide a model that significantly improves upon currently available approaches, allowing us to move forward our investigations. The Trust’s funding has been invaluable allowing us to develop this model.
APRIL 2013 – THE START NEW
Support from the Trust has been invaluable in providing the unique opportunity to appoint a Humane Research Trust Postdoctoral Research Assistant, Dr Charlotte Green, to develop a new theme of research in my laboratory. I typically investigate liver metabolism in human subjects with the use of non-radioactive labelled ‘tracers’, but this approach does not provide specific information about the metabolic processes that are happening within the liver cells (hepatocytes). Dr Green will work with myself, Dr Karl Morten (Mitochondrial Metabolism, University of Oxford) and Mr Michael Silva (Liver Surgeon, Oxford University Hospitals NHS Trust) to develop a model using human hepatocytes that allows us to study why it is some individuals start to get an accumulation of fat in their liver cells. Liver fat accumulation (hepatic steatosis) is associated with excess fat (triglyceride) in the blood, and this is a risk factor for heart disease (CVD) and diabetes. The reason why the liver starts to accumulate fat is poorly understood at this point in time, but understanding the mechanisms by which the liver accumulates fat will provide further insight into pathways that could be targets to alter liver fat metabolism to lower the risk of CVD and diabetes.
Due to the shortage of donor organs, transplant centres are increasingly turning to livers that would once have been discarded – ‘marginal’ organs, such as those with steatosis and use of these organs could decrease the likelihood of a successful transplant. The mechanisms by which steatosis causes post-transplant complications are not fully understood.
The amount of fat in hepatocytes is regulated by a number of distinct mechanisms. Previous studies of these processes have largely been performed in animals or cells derived from animals with results difficult to translate directly to humans due to differences in liver metabolism and enymes between species. We are aiming to develop a model that uses excess liver tissue (resection surplus) that is normally discarded from previously consented patients undergoing liver surgery to isolate human hepatocytes which can then be used to understand the mechanisms of hepatic steatosis (Figure 1). Human hepatocytes can be difficult to isolate from intact liver tissue, be fussy about the environment they are cultured in and can, at times, be difficult to work with. We will be developing different approaches for isolating hepatocytes from liver tissue to produce cells with a good yield and long-term viability. We hope culturing the hepatocytes under different conditions, in order to mimic their environment inside the body; will produce a model which significantly improves upon approaches currently available.
This project will help develop and validate a model that will then pave the way to investigations of the mechanisms that regulate liver fat metabolism as well as provide knowledge of how specific environmental and lifestyle factors (such as fats, sugars and oxygen concentration) influence the regulation of liver fat. Ultimately we want to also establish a system where we can store hepatocytes long term in liquid nitrogen, a process known as cryopreservation. Cryopreservation will be designed to retain liver function and health of the hepatocytes allowing us to make them a valuable commodity for the wider research community.