Modelling sex differences in metabolic dysfunction-associated steatotic liver disease using stem cell-derived liver tissue

Abstract

Liver disease claims around 2 million lives annually, with liver cirrhosis and hepatocellular carcinoma being the most common causes of death. The most prevalent factors when developing cirrhosis are extensive alcohol use, chronic hepatitis and metabolic dysfunction-associated steatotic liver disease (MASLD). MASLD is an umbrella term for a variety of different disorders with ranging severity of liver inflammation and fibrosis. Such disorders include a fatty liver (steatosis) which is associated with F0/F1 stage fibrosis. In a fraction of patients, MASLD develops into inflammatory disease, termed metabolic dysfunction-associated steatohepatitis (MASH). This is associated with moderate fibrosis (F2) in the early stages. MASH then progresses in subset of patients (F3 fibrosis) and may culminate in cirrhosis (F4 fibrosis) and hepatocellular carcinoma. MASLD is a metabolic disorder, highly influenced by other diseases such as type II diabetes (T2D), cardiovascular disease and obesity. Despite current MASLD prevalence being over 30%, there is currently only one drug available, with many candidate drugs failing in the late phases of clinical trials. This highlights the need for a better understanding of the disease in order to improve drug development and medical intervention. Liver homeostasis is influenced by a number of factors, including sex hormones, in particular testosterone and estrogen. Men are twice as likely to die from liver disease or develop MASLD than women. However, women post-menopause have the same risk as men, suggesting that the metabolic age influences the MASLD establishment and disease progression. Clinical and animal studies demonstrated that balanced concentrations of estrogen and testosterone in healthy individuals reduce the risk of impaired glucose and fat metabolism. Therefore, hormone replacement therapy (HRT) could be a viable therapy. Indeed, HRT has been shown to reduce the development of T2D in men and women. However, it is not a suitable treatment option for all patients and has risks associated with its long term usage. My research project focused on developing a better understanding of sex hormone signalling in metabolic liver disease. I employed human pluripotent stem cells (PSCs) as a renewable resource to generate male and female liver tissue. Stem cell derived liver tissue was composed of hepatocytes, endothelial and stellate cells which are important in health and disease. I hypothesised that in vitro engineered liver tissue could effectively model sex hormone signalling in MASLD. Following biochemical confirmation of metabolic dysfunction, two transcriptomic approaches (NanoString and single nuclei RNA-sequencing) were employed to better understand the protective roles played by sex hormone signalling in disease development. Those results were benchmarked against RNA-sequencing from a human patient database, SteatoSITE, encompassing a full spectrum of MASLD and MASH in humans. The protective mechanisms of testosterone and estrogen exposure were uncovered at a gene expression level. Although gene expression differences were observed between sexes, the pathways important for prevention against late-stage MASH were similar between male and female liver spheres. In conclusion, the novel findings of my thesis identified new patterns of gene regulation in human metabolic liver disease, with several candidate targets already druggable. Moving beyond these studies, the platform described in my study could be used to model other liver diseases, and to develop new biomarkers and precision medicines of the future.