iPSC-Derived Hepatocytes from Patients with MASLD Exhibit Early Mitochondrial Dysfunction

Background & Aims A hallmark of metabolic dysfunction-associated steatotic liver disease (MASLD) is a decline in the ability of hepatocyte mitochondria to adapt to excess lipid. This leads to the production of reactive oxygen species (ROS) and the instigation of a vicious cycle of further mitochondrial damage and cellular dysfunction that promotes disease progression. In this study, we investigated whether induced pluripotent stem cells (iPSCs) from MASLD patients exhibit features of mitochondrial dysfunction when differentiated to hepatocyte-like cells (iPSC-Heps). Methods iPSCs from 10 MASLD patients and 10 healthy control subjects genotyped for the I148M variant of PNPLA3 were differentiated to iPSC-Heps. Mitochondrial mass and function were assessed under basal culture conditions and following short-term exposure to exogenous palmitate. Outcomes included gene expression, mitochondrial oxygen consumption, ROS production and cellular energy status. Results iPSC-Heps from MASLD patients spontaneously accrued more lipid than control iPSC-Heps. Mitochondrial content was similar in MASLD and control iPSC-Heps, but MASLD iPSC-Heps displayed significant differences in mitochondrial function including a decrease in oxygen consumption rate when challenged with palmitate. Antioxidant gene expression was increased at baseline in MASLD vs. control iPSC-Heps, and MASLD iPSC-Heps produced more ROS and less ATP than controls after palmitate treatment. Differences persisted even when controlling for PNPLA3 genotype. Conclusions iPSC-Heps from MASLD patients exhibit mitochondrial alterations characteristic of their diseased origin. The degree of mitochondrial dysfunction seen in MASLD iPSC-Heps is reminiscent of that described clinically in early MASLD, prior to progression to steatohepatitis. Mitochondrial alterations in MASLD iPSC-Heps occur independently of PNPLA3 genotype.