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Title: Induced pluripotent stem cell derived liver model for the study of PNPLA3-associated non-alcoholic fatty liver disease
Author: Tilson, Samantha Grace
ISNI:       0000 0004 9359 9875
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2020
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Non-alcoholic fatty liver disease (NAFLD) is now the leading cause of chronic liver disease in the developed world, afflicting approximately one in four adults globally. NAFLD is defined by the accumulation of fat within the liver which ranges in severity from simple steatosis to non-alcoholic steatohepatitis (NASH), cirrhosis, and hepatocellular carcinoma. Until recently, NAFLD has been considered to be a consequence of metabolic syndrome; however, recent studies suggest that genetic factors may also influence disease onset and progression. Accordingly, genome wide association studies have linked the I148M variant in the gene coding for Patatin-like phospholipase domain-containing protein 3 (PNPLA3) with NAFLD aggravation without underlying metabolic disease. However, despite over a decade of research, the function of PNPLA3 and its role in the pathogenesis of NAFLD remains largely obscure. The lack of clarity regarding the function and disease associations of PNPLA3 is due in large part to the lack of a comprehensive human model of PNPLA3-associated NAFLD. In order to address this need, we have developed an in vitro model that takes advantage of the unique properties of human induced pluripotent stem cells (hiPSC) and the CRISPR/CAS9 gene editing technology. We first used CRISPR/CAS9 to generate hiPSC lines with either a knock-out (PNPLA3KO) of the PNPLA3 gene or with the I148M variant knocked in (PNPLA3I148M). The resulting cells were then differentiated into hepatocytes and grown in 3D culture conditions to improve their maturity and functionality. The differentiated cells were treated with either monounsaturated or saturated free fatty acids to induce NAFLD-like phenotypes (lipid accumulation and lipid toxicity, respectively) and characterized by various functional, genomic, and lipidomic assays. The genetically edited sublines showed similar differentiation efficiency toward hepatocytes as untargeted cells indicating that changes in PNPLA3 activity does not affect hepatic development. Following treatment with free fatty acids, PNPLA3KO cells showed higher lipid accumulation than untargeted cells as well as an altered pattern of response to lipid-induced stress. Indeed, PNPLA3KO cells were resistant to saturated fatty acid-induced lipotoxicity. Furthermore, lipidomic analyses suggest that the PNPLA3 edited cells may be avoiding cell death by shuttling the saturated fatty acids into triglycerides rather than other metabolic pathways. These findings were initially incongruent with the human disease; however, despite their resistance to lipid induced stress, the PNPLA3KO cells downregulated all phases of drug metabolizing enzymes which made them more susceptible to other forms of hepatotoxicity such as ethanol. The PNPLA3I148M cells exhibited an intermediate phenotype between untargeted and PNPLA3KO cells. These results demonstrate for the first time in a fully human model that the I148M variant in PNPLA3 is a loss of function variant. This loss of PNPLA3 activity leads to the global downregulation of metabolic pathways likely due to the sequestration of fatty acids in triglycerides caused by reduced lipid droplet remodelling capacity in these hepatocytes. Our results indicate that patients carrying the I148M variant have lower hepatic metabolic activity which causes steatosis, reduced susceptibility to lipotoxicity, and increased susceptibility to other forms of hepatotoxicity which may contribute to NAFLD progression. This novel system provides the first opportunity to study the role of PNPLA3 in the development and progression of human NAFLD in vitro.
Supervisor: Vallier, Ludovic ; Liang, T. Jake Sponsor: National Institutes of Health ; National Institute of Diabetes and Digestive and Kidney Diseases ; Wellcome Trust ; MRC ; Cambridge Stem Cell Institute ; International Biomedical Research Alliance ; European Research Council
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
Keywords: iPSC ; NAFLD ; PNPLA3 ; in vitro disease modelling ; hepatology