Navigating liver disease complexity: Innovations in in vitro modeling and precision medicine
Authors: Amaya Lopez-Pascual
Journal: Hepatology (2023)

Chronic liver disease (CLD) manifests as a gradual decline in liver functions, persisting for over 6 months and may involve the impairment of vital processes such as clotting factor synthesis, protein production, detoxification, and bile excretion. Etiologically diverse CLD arises from factors including exposure to toxins, prolonged alcohol abuse, infections, autoimmune disorders, and genetic and metabolic disorders. CLD is characterized by a continuous cycle of inflammation, parenchymal damage, and regeneration, ultimately leading to the development of fibrosis and cirrhosis.1 The cellular mechanisms underpinning fibrosis and cirrhosis involve the recruitment of stellate cells and fibroblasts, culminating in fibrotic tissue formation, while the regeneration of liver parenchyma relies on the activity of hepatic stem cells. Cirrhosis manifests as a profound disruption in liver architecture, marked by the formation of extensive nodules, vascular restructuring, neoangiogenesis, and the accumulation of an extracellular matrix. Cirrhosis is an irreversible stage or end-stage liver disease occurring when the liver is damaged beyond repair. Hepatotropic viral infections—HBV and HCV—alcohol-associated liver disease, and NAFLD—recently renamed as metabolic dysfunction–associated steatotic liver disease—are the main leading causes of CLD.1,2 These liver injuries eventually contribute to the progression toward HCC, which is a major cause of cancer-related deaths worldwide.2 Given its widespread prevalence, understanding the common etiologies, clinical manifestations, and effective management of CLD is paramount. In vitro two-dimensional (2D) models do not fully capture the hepatic microenvironment of human chronic diseases, and large animal models are expensive and logistically difficult to use. Small animals such as rodents have been extensively used; however, they often do not fully reproduce the hallmarks of human CLD, and thus have drawbacks when used for novel therapies for the human disease. Frequently, innovative therapies that prove effective in rodent studies encounter setbacks during preclinical and early clinical trials, often attributed to drug toxicity or ineffectiveness. Further, approximately 50% of drugs are withdrawn or discontinued in post-approval actions after causing adverse effects to a significant proportion of the population.3 Thus, alternative models recapitulating the hepatic microenvironment in CLDs are warranted. Over the decades, traditional immortalized cell cultures (2D models) derived from HCCs, either in monoculture or co-culture with other liver resident cells, have been broadly used as state-of-the-art in vitro models for the study of CLDs. In this sense, recent advances in the development of accurate systems modeling human tissues, e.g. spheroids, organoids, induced pluripotent stem cells, (iPSCs) and organ-on-chip have shown great potential for the study of the multifaceted aspects of human pathophysiology, shedding light on its intricate mechanisms, clinical implications, and therapeutic considerations. While these models are not as commonly employed as 2D cultures, there is a growing trend in their utilization to optimize CLD models. In an effort to address these hurdles, some researchers have successfully developed iPSC-derived or cell-line–derived organoid models for the study of CLDs.4–7 This innovative approach provides insights that extend beyond the scope of traditional 2D cultures. However, the preservation of the authentic proportions between parenchymal and nonparenchymal cells found in the liver in these multicellular liver organoid models is complicated. In the quest to unravel the intricate mechanisms of CLD, the study conducted by Lee et al8 published in this issue of Hepatology represents a significant stride in the field, particularly in the realms of HCV infection and NAFLD progression models. This work introduces a novel multicellular liver organoid model closely mimicking the cellular composition and architecture of the human liver that transcends the limitations of conventional 2D cell cultures. Compared to previously published studies using similar in vitro systems, this model offers a more holistic understanding of the liver cell populations in the complex interplay between viral infections and metabolic liver disorders. A previous work performed by this group4 reported the development of iPSC-derived hepatic models capable of long-term expansion with competent liver functionality, that is, serum protein production, drug metabolism and detoxifying functions, active mitochondrial bioenergetics, and regenerative and inflammatory responses. Now, Lee et al8 ingeniously harnessed the potential of iPSCs to craft a multicellular liver organoid model, providing an unprecedented platform for investigating the dynamic processes associated with HCV infection and NAFLD. What distinguishes this study is its meticulous incorporation of patient-specific iPSCs differentiated into hepatocytes, cholangiocytes, stellate cells, and Kupffer-like macrophages, capable of responding to drugs with proven efficacy in patients, thereby reflecting individual genetic predispositions in CLD manifestation. In the context of HCV infection, the liver organoid model presented a three-dimensional (3D) landscape that intricately mimics the in vivo microenvironment. Through this lens, Lee et al8 delved into the dynamics of viral replication, changes in cellular architecture, and the orchestration of the host immune response. Remarkably, this model serves a dual purpose by offering a unique opportunity to probe into the complexities of NAFLD progression. The organoids exhibited hallmarks of NAFLD, such as lipid accumulation and alterations in gene expression associated with metabolic dysfunction. This dual functionality of iPSC-derived liver organoids provides a comprehensive view of CLDs, allowing researchers to explore the cross talk between viral infections and metabolic disorders, a facet that has remained challenging to decipher in previous models. Besides, this model showed great potential as an evaluation platform for new NASH and HCV drugs, which holds promise for enhanced predictive accuracy in future drug screening studies. While acknowledging the remarkable contributions made by Lee et al8, it is essential to appreciate the collective advance in CLD models. Several works highlight the potential of novel 3D platforms to study the relevance of individual genetic backgrounds in NAFLD progression and genetic CLDs.6,7,9,10 These studies—briefly mentioned hereafter—with their respective in vitro 3D liver systems complement the landscape recently created by Lee and colleagues. However, the potential of liver organoid and iPSC models lies not only in deciphering disease mechanisms but also in revolutionizing drug development and high-throughput screening. Tilson et al6 investigated the impact of the PNPLA3 I148M variant on NAFLD. These authors used CRISPR-Cas9 genetically engineered iPSCs differentiated to hepatocyte organoids to successfully demonstrate that changes in PNPLA3 activity influenced the metabolism of human hepatocytes, providing a novel perspective of how specific genetic elements contribute to disease phenotypes. In line with the genetic variability associated with CLD, Guan et al9 also analyzed genetic diseases using CRISPR-engineered hepatic organoids derived from iPSCs. Their groundbreaking work uncovered direct links between various genetic liver disorders and human pathophysiology, enhancing the translational relevance of this model in the development of targeted therapies for genetic liver diseases. Crouchet et al7 took the organoid model to the next level by using a human liver cell line–based organoid system for developing a clinical prognostic liver signature and validated their model in rodent and patient-derived primary hepatocytes. This study incorporated state-of-the-art techniques to model their signature for therapeutic discovery, enhancing their ability to predict CLD progression toward HCC and bringing an essential clinical perspective with the opportunity to tailor personalized interventions (Figure 1). More recently, Bronsard et al10 employed 3D multi-cell-type liver organoids specifically designed for drug safety assessments in NAFLD. Their innovative multicellular approach incorporated cell lines relevant to NAFLD pathology, providing a robust platform for drug development in a more physiologically relevant context. On the other hand, Nie et al5 used 3D cultures for the study of hepatotropic viral infections. Focused on HBV-host interactions using iPSC-derived liver organoids, they uncovered key aspects of the host-virus interactions, providing valuable information for developing antiviral strategies. This study's findings contribute to our understanding of viral infections in the liver and offer potential targets for therapeutic intervention.FIGURE 1: Cell sources for liver in vitro models and applications in translational research and medicine. Liver cells are obtained either from liver tissue or from established cell lines. Primary cultures isolated from human liver samples can be separated into the primary human hepatocyte fraction, the primary human nonparenchymal cells and the pluripotent or multipotent stem cell fractions (PSCs and MSCs, respectively). Immortalized cell lines, resembling diverse liver cell types, are predominantly characterized by cancer cell line features. These cell lines offer advantages in terms of simplicity, cost-effectiveness, and reproducibility compared to liver primary cells and stem cells. All these cell types can be employed to create 2D and 3D models. 2D cultures are characterized by single or mixed cell types in monolayer co-cultures. In contrast, 3D models range from simpler forms, where disaggregated cells self-aggregate into spheroids containing at least one cell type, to more intricate liver organoids exhibiting cellular composition and architecture similar to the liver. Emerging technologies such as organ-on-a-chip systems represent the cutting edge in this spectrum. The technical complexity, economic cost, representativeness of the liver, accuracy in reproducing disease phenotypes, and the system's lifespan increase across these models. Applications of these models span from studying liver function to investigating the pathophysiology of liver diseases. Moreover, they prove valuable in drug discovery screenings, high-throughput studies, and omics analyses, all holding high translational potential. At a more clinical level, these models facilitate regenerative medicine applications by enabling the differentiation and assembly of liver buds for transplantation. Additionally, they contribute to therapy personalization through the study of the genetic background, allowing the tailoring of drug therapies. Abbreviations: 2D, two dimensional; 3D, three dimensional; MSCs, multipotent stem cells; PSCs, pluripotent stem cells.Each study's strengths lie in its unique approach and model system. All studies, however, acknowledge the challenges of cohort size, standardization, and the in vitro nature of their respective models. As these models grow in complexity and representativeness, they also increase their economic and infrastructural costs and may not be readily accessible to all research groups. These resource-intensive needs limit the widespread adoption of such models in different settings. On top of that, despite proposing a valuable tool for studying genetic variants offering a patient-specific perspective, organoids could be oversimplified paradigms that may not fully recapitulate the complexity of the adult human liver. In essence, the consolidation of advanced technologies, the generation of patient-specific iPSCs, and a comprehensive approach to modeling CLDs position the study by Lee and colleagues into the forefront of hepatic research models. The fidelity in mimicking liver microenvironment is especially critical when studying CLDs with multifaceted etiologies, where the interplay between different cell types plays a pivotal role in disease progression. While iPSC-based models have proven invaluable in disease modeling, the use of human primary liver organoids introduces a distinct advantage by capturing the inherent complexity and heterogeneity of the liver. The personalized approach of using iPSCs to preserve the individual genetic background enables a more comprehensive understanding of how genetic factors contribute to disease phenotypes, offering a path toward precision medicine in CLD research. The incorporation of liver organoids derived from cell lines or iPSCs into drug development pipelines holds promise for accelerating the discovery and validation of novel treatments for liver diseases, heralding a new era in precision medicine. This is particularly crucial given the limitations associated with animal models, which often fail to accurately predict human drug metabolism and toxicity. As we navigate the landscape of liver research, the integration of these organoid models stands as a transformative leap toward more accurate disease modeling, drug discovery, ultimately bringing us closer to improved clinical outcomes for patients with CLDs.