Construction of liver organoid models by hepatobiliary differentiation from human induced pluripotent stem cells: state of the art, challenges and improving strategies

Abstract

Physiologically relevant liver models are essential for advancing hepatic disorder research, especially for disease modeling and drug development, yet current in vitro systems fail to adequately recapitulate the architecture and function of the liver. Owing to the accessibility, robust proliferation and multilineage differentiation potential of human induced pluripotent stem cells (iPSCs), liver organoids derived from iPSCs have emerged as a promising resource in hepatology. Despite this promise, the field still faces persistent bottlenecks including incomplete hepatic maturation, insufficient incorporation of non-parenchymal cells (notably immune and stromal populations), phenotypic instability, and a lack of consensus on standardized differentiation protocols. Therefore, this review systematically analyzes the challenges and strategies of iPSC differentiation into liver organoids and the related influencing factors by focusing on multidimensional regulation of hepatobiliary development as well as the effects of cellular origin, culture system and liver microenvironment on hepatic differentiation of iPSCs. Moving forward, priority should be given to the following directions: (1) Elucidating the self-assembly mechanism of liver organoids to enable precise control of hepatobiliary differentiation, thereby better governing organoid morphology and improving reproducibility; (2) Replacing exogenous cytokines with small-molecule compounds at different stages of iPSC differentiation to simplify and standardize differentiation protocols; (3) Advancing liver organoid transplantation as a means to validate physiological functionality and shift cell therapy from passive replacement toward active tissue reconstruction; (4) Integrating artificial intelligence to achieve intelligent and precise regulation of hepatic differentiation.

1. Introduction

The liver is a core metabolic organ integrating diverse physiological functions including detoxification, drug metabolism and protein synthesis etc. However, it is prone to injury and dysfunction caused by various factors such as viral infections, toxins and autoimmunity [1]. Current statistics indicates that approximately two million deaths annually are attributed to liver diseases worldwide, thus highlighting the urgent need to improve the treatment of liver diseases [2]. Developing physiologically relevant liver models is essential for hepatic disorder research, yet accurately recapitulating human hepatic physiology remains difficult due to the liver's complex cellular composition and its dynamic interactions with other organs, such as the gastrointestinal tract and pancreas [1, 3]. Liver organoids, as three-dimensional cell clusters mimicking the structure and function of native organ, serve as promising new models for mechanistic studies and drug evaluation [4]. Among construction methods, induced pluripotent stem cells (iPSCs) have emerged as the most promising alternative for their potential to offer individual-specific liver cells while circumventing limitations of other models, such as donor variability and limited availability of primary hepatocytes [5, 6].

However, current liver organoid construction still faces many challenges in recapitulating the structure and function of native human liver. For instance, existing organoids frequently exhibit incomplete hepatic maturation and phenotypic instability, and their differentiation process remains complex, with varying protocols yielding either a heterogeneous population of hepatocyte-like and cholangiocyte-like cells or exclusively hepatocyte-like cells (HLCs), thereby introducing limitations and uncertainties in application [7]. Additionally, most liver organoids lack non-parenchymal cells, particularly stromal and immune components. During early liver development, hepatic mesenchymal cells comprise diverse subpopulations with distinct supportive and regulatory functions [8]. Yet current models either include only a single subtype or omit mesenchymal elements entirely, thereby failing to recapitulate the native mesenchymal niche [9]. The incorporation of immune components also remains at a preliminary stage, although they play crucial roles in both disease modeling and the low-grade inflammatory milieu that shapes early hepatic organogenesis [10].