Modeling pathogenesis and progression of metabolic dysfunction-associated steatotic liver disease and therapeutic drug screening using hESC-derived mature polarized hepatocyte organoids

Abstract

Background: Metabolic dysfunction-associated steatotic liver disease (MASLD), the most prevalent chronic liver disorder worldwide, exhibits complex pathogenesis and lacks effective targeted therapeutics. Existing animal models are limited by prolonged induction periods and interspecies discrepancies, while conventional monolayer hepatocyte cultures fail to recapitulate disease pathology due to inadequate polarization and functional immaturity. Methods: To overcome these limitations, we established an in vitro MASLD model by treating human embryonic stem cell (hESC)-derived mature polarized hepatocyte organoids (P-hep-orgs) with free fatty acids (FFAs). Pathogenesis and progression of MASLD in this model were characterized using multiple assays, and its utility for drug screening was validated with three known antioxidant or lipid-lowering agents. Results: P-hep-orgs derived from hESCs expressed mature hepatocyte markers (e.g., ALB), exhibited polarized architecture (e.g., MRP2) and demonstrated functionalities of mature hepatocytes (e.g., urea production). Moreover, we developed an in vitro MASLD model by treating P-hep-orgs with FFAs. This model recapitulated key pathological progression hallmarks, including disrupted glucose/lipid metabolism, oxidative stress, apoptosis, loss of polarization, impaired liver function, and ductular reaction. Furthermore, transcriptomic analysis revealed that P-hep-orgs treated with FFAs for 10 days shared similar molecular signatures with human MASH liver tissues (581 overlap DEGs). Finally, this model was used to assess the potential efficacy of established antioxidant or lipid-lowering agents (e.g., Vitamin E) in alleviating pathological phenotypes, including lipid accumulation and oxidative stress.

1. Introduction

Metabolic dysfunction-associated steatotic liver disease (MASLD), formerly termed non-alcoholic fatty liver disease (NAFLD), represents the most prevalent chronic liver condition globally, affecting approximately 30% of the population [1]. As a leading etiology of liver-related morbidity and mortality, including cirrhosis and hepatocellular carcinoma [2, 3], MASLD poses a significant clinical burden. Its pathogenesis involves multifactorial mechanisms [4], and there exists no specific drug currently [5, 6]. These challenges underscore the urgent need for physiologically relevant models that accurately recapitulate disease progression to facilitate mechanistic studies and therapeutic discovery.

Current research on MASLD predominantly employs animal models such as mice [7] and rats [8] to study MASLD, yet these studies suffer from protracted induction times, inter-batch variability, and interspecies physiological discrepancies [9]. While in vitro cell models mitigate some limitations of animal studies, conventional systems inadequately replicate the structural and functional complexity of human hepatocytes, hindering breakthrough research on MASLD. Hepatocyte polarization underpins critical physiological functions, such as metabolism, secretion, and detoxification [10]. In vivo, hepatocytes establish two functional domains through the asymmetric distribution of membrane proteins (e.g., Na+/K+-ATPase, bile acid transporters), which demarcate the basolateral domain adjacent to hepatic sinusoids and the apical membrane domain facing bile canaliculi. This polarized configuration regulates the directional uptake and secretion of carbohydrates, lipids, and other molecules [11], thereby governing their absorption, transport, and metabolic pathways. The disruption of hepatocyte polarization induces abnormal lipid metabolism, thereby enhancing lipid toxicity to hepatocytes. Existing MASLD cell models rely primarily on either immortalized hepatocyte cell lines (e.g., HuH7, HepaRG) [12, 13] or two-dimensional (2D) hepatocytes derived from human pluripotent stem cells (hPSCs) [14, 15]. Alternatively, these hepatocytes are enzymatically dissociated into single cells, losing their polarized architecture and functional maturity.