ABSTRACT/SUMMARY Our long-term goal remains the same and targets to develop human liver tissue model from stem cells, with multi-cellular cues, metabolic functionality, that incorporates dynamic genetic and epigenetic factors and encompasses the spectrum and evolution of human NAFLD to uncover disease mechanism and therapeutics. The objectives of the proposed study are to continue developing human fatty livers engineered with iPS cells, that incorporates the dynamic expression of the epigenetic-related histone modification SIRT1 in different genetic polymorphisms and elucidate their role as metabolic regulators in the development of human fatty liver disease. The central hypothesis to be tested here is that genetic polymorphisms especially the NAFLD-high- risk PNPLA3 rs738409, combined with epigenetic-related changes (SIRT1) can control hepatic metabolism and contribute to the development of human fatty liver. The rationale is that the ability to generate human diseased liver tissue using genetically modifiable iPS cells from different human populations with single- nucleotide-polymorphisms is a powerful resource, that now make it possible, for the first time, to functionally interrogate their role in disease. Importantly, based on our strong preliminary data of targeted metabolomic analysis in isolated human hepatocytes obtained from either liver resections “Normal Human Hepatocytes” or hepatocytes isolated from explanted NASH livers “NASH Human Hepatocytes” carrying either the NAFLD-high risk PNPLA3 I148M genetic variant or wild type we hypothesize that PNPLA3 I148M hepatocytes have high levels of ferroptosis thereby leading to low metabolic capacity to adapt to stressors and continuous loss of hepatocytes. In the newly proposed aim3, we aim to uncover ferroptosis based therapeutic strategies for patients with NAFLD-high risk PNPLA3 I148M genetic variants. The work described here is expected to i) generate human iPS cells carrying shRNA mediated conditional knockdown of SIRT1 in different genetic backgrounds (especially the NAFLD-high-risk PNPLA3 rs738409 variant), ii) develop a novel approach for modeling an organ-like environment to determine the role of the epigenetic related factor SIRT1 and the genetic variants in the development of fibrotic human mini-livers with fatty liver disease and iii) determine the role of genetic polymorphisms in regulating functional metabolism in mini-human iPS-derived liver tissue with fatty liver disease with especial focus on ferroptosis. Understanding this process alone or in relation to epigenetic changes in a human liver tissue model can result in preventive therapeutic strategies for patients with NAFLD-high risk PNPLA3 I148M genetic variants. The results of this work will also have a positive impact by establishing the basis and platform for future sophisticated organ engineering techniques that incorporates several different cell types from the same iPS cell source, these techniques could be applied to ...