Development of an alcoholic liver disease model for drug evaluation from human induced pluripotent stem cell-derived liver organoids

Abstract

Alcoholic liver disease (ALD) poses a significant health challenge, so comprehensive research efforts to improve our understanding and treatment strategies are needed. However, the development of effective treatments is hindered by the limitation of existing liver disease models. Liver organoids, characterized by their cellular complexity and three-dimensional (3D) tissue structure closely resembling the human liver, hold promise as ideal models for liver disease research. In this study, we use a meticulously designed protocol involving the differentiation of human induced pluripotent stem cells (hiPSCs) into liver organoids. This process incorporates a precise combination of cytokines and small molecule compounds within a 3D culture system to guide the differentiation process. Subsequently, these differentiated liver organoids are subject to ethanol treatment to induce ALD, thus establishing a disease model. A rigorous assessment through a series of experiments reveals that this model partially recapitulates key pathological features observed in clinical ALD, including cellular mitochondrial damage, elevated cellular reactive oxygen species (ROS) levels, fatty liver, and hepatocyte necrosis. In addition, this model offers potential use in screening drugs for ALD treatment. Overall, the liver organoid model of ALD, which is derived from hiPSC differentiation, has emerged as an invaluable platform for advancing our understanding and management of ALD in clinical settings.

1. Introduction

A report from the World Health Organization indicated that alcohol abuse led to more than 3 million deaths in 2018, accounting for 1 in 20 deaths and contributing to more than 5% of the global disease burden. Prolonged alcohol abuse can lead to the development of alcoholic liver disease (ALD), which encompasses fatty liver disease (steatosis), alcoholic hepatitis, and alcoholic cirrhosis [1,2]. The pathological features of ALD include hepatocyte steatosis, oxidative stress, inflammation, and liver fibrosis. Disease progression can lead to hepatocyte necrosis and apoptosis, liver cirrhosis, and even liver cancer [3,4]. At present, the models used to study ALD mainly include animal models and liver cell line culture models [5,6]. Two-dimensional cultures of liver cell lines have traditionally been used to mimic diseases and develop medications. For instance, flat cultures of liver stellate cells (HSCs) have been employed, with or without the addition of soft hydrogels such as polyacrylamide gels. However, the genetic profiles of these cells often do not align with those of cirrhotic tissues in humans. Translating findings from 2D cultures has been challenging due to their inability to replicate natural characteristics such as dynamic physical and chemical signals and the microenvironmental structures present in the liver lobule, which often leads to a rapid decline in liver cell function [7]. Therefore, the application of these models in ALD research is limited due to species differences and the constraints of two-dimensional (2D) culture [8–10].

Organoids refer to the self-assembly of adult stem cells or pluripotent stem cells in a 3D in vitro culture environment to form tissue analogues with 3D structures that accurately reflect the characteristics of the original tissue (derived from adult stem cells) or directed differentiated tissue (derived from induced pluripotent stem cells) [11]. Organoids originated from iPSCs are widely recognized as pivotal elements in the field of disease modelling based on organoids. Recent studies have demonstrated the successful development of vascularized organoids derived from iPSCs. In a specific study, the integration of stromal elements such as vasculature, fibroblasts, and immune cells was achieved by utilizing mesoderm progenitor cells induced from iPSCs [12]. Organoid culture offers the potential for long-term in vitro culture expansion while maintaining stable genetic characteristics, thus providing a solution to the challenges of liver disease models [13]. In vitro organoid models represent a significant advancement for translational research, as these 3D models closely mimic in vivo biological processes such as tissue renewal and the response of tissues to drugs, toxins, and mutagenesis. Compared to traditional monolayer cultures, 3D liver organoid cultures offer more accurate models for studying hepatic toxicity.

In the present study, we initially differentiated ...