MODELLING HORMONAL REGULATION OF NAFLD AND NASH USING STEM CELL DERIVED LIVER SPHERES

Introduction

Liver disease claims 2 million lives annually, with cirrhosis being the most common cause of death (Asrani et al., 2019). With the increasing span of life and better economic conditions, more adults suffer from diabetes and obesity, which are risk factors for development and progression of non-alcoholic fatty liver disease (NAFLD) (Sookoian & Pirola, 2019). As the liver is a highly metabolic organ, chronic disruption of its metabolic function can have severe consequences, including disease progression to cirrhosis and hepatocellular carcinoma (Kanwar & Kowdley, 2016; Asrani et al., 2019). Liver function is regulated by a number of factors, including hormonal signalling (Shen and Shi, 2015). Oestradiol increases lipolysis, cholesterol secretion and catabolism of glucose, whilst decreasing the accumulation of fatty acids (Shen and Shi, 2015). In contrast, testosterone enhances glycogen synthesis, expression of the insulin receptor and cholesterol storage (Shen and Shi, 2015). Previous studies showed that post-menopausal women have an increased risk of insulin resistance, visceral fat accumulation, cardiovascular disease and hyperlipidaemia (Phan and Toth, 2014). Before the menopause, NAFLD prevalence is higher in men than women.

Methods

We have developed male and female in vitro human liver models from pluripotent stem cells. We have previously used them to understand metabolic alterations in hepatocyte function in 2D culture following the development of macrovesicular steatosis (Sinton et al., 2021). Since those studies we have developed 3D liver tissue platforms and used them to better understand the effect of sex hormone signalling on the development of steatosis. Liver sphere gene expression was examined using Nanostring technology and compared to a human bulk hepatic RNA-seq database covering the full spectrum of human NAFLD (https://steatosite.com/).

Results

Liver spheres consisting of hepatocytes, endothelial cells and stellate cells were generated by cell self-assembly as previously described and display characteristic hepatocyte function (Lucendo-Villarin et al., 2021). Subsequently, liver spheres exposed to a high energy substrate diet developed steatosis and displayed differential gene expression in response to specific hormonal stimuli. Changes in the transcriptomic signatures of liver spheres, defined by Nanostring, were then compared to the SteatoSITE database.

Conclusion

Stem cell-based liver spheres were able to accurately model human NAFLD and were also predictive of disease progression in both male and females. Following further analysis, the validated and novel gene signatures identified in these studies will be presented at the meeting.