The liver is a very complex organ that ensures numerous functions; it is thus susceptible to multiple types of damage and dysfunction. Since 1983, orthotopic liver transplantation (OLT) has been considered the only medical solution available to patients when most of their liver function is lost. Unfortunately, the number of patients waiting for OLT is worryingly increasing, and extracorporeal liver support devices are not yet able to counteract the problem. In this review, the current and expected methodologies in liver regeneration are briefly analyzed. In particular, human pluripotent stem cells (hPSCs) as a source of hepatic cells for liver therapy and regeneration are discussed. Principles of hPSC differentiation into hepatocytes are explored, along with the current limitations that have led to the development of 3D culture systems and organoid production. Expected applications of these organoids are discussed with particular attention paid to bio artificial liver (BAL) devices and liver bio-fabrication.

Liver transplantation is currently the only curative treatment for several liver diseases such as acute liver failure, end‐stage liver disorders, primary liver cancers, and certain genetic conditions. Unfortunately, despite improvements to transplantation techniques, including live donor transplantation, the number of organs available remains insufficient to meet patient needs. Hepatocyte transplantation has enabled some encouraging results as an alternative to organ transplantation, but primary hepatocytes are little available and cannot be amplified using traditional two‐dimensional culture systems. Indeed, although recent studies have tended to show that three‐dimensional culture enables long‐term hepatocyte culture, it is still agreed that, like most adult primary cell types, hepatocytes remain refractory to in vitro expansion. Because of their exceptional properties, human pluripotent stem cells (hPSCs) can be amplified indefinitely and differentiated into any cell type, including liver cells. While many teams have worked on hepatocyte differentiation, there has been a consensus that cells obtained after hPSC differentiation have more fetal than adult hepatocyte characteristics. New technologies have been used to improve the differentiation process in recent years. This review discusses the technical improvements made to hepatocyte differentiation protocols and the clinical approaches developed to date and anticipated in the near future.

Familial hypercholesterolemia type IIA (FH) is due to mutations in the low-density lipoprotein receptor (LDLR) resulting in elevated levels of low-density lipoprotein cholesterol (LDL-c) in plasma and in premature cardiovascular diseases. As hepatocytes are the only cells capable of metabolizing cholesterol, they are therefore the target cells for cell/gene therapy approaches in the treatment of lipid metabolism disorders. Furthermore, the LDLR has been reported to be involved in hepatitis C virus (HCV) entry into hepatocytes; however, its role in the virus infection cycle is still disputed. We generated induced pluripotent stem cells (iPSCs) from a homozygous LDLR-null FH-patient (FH-iPSCs). We constructed a correction cassette bearing LDLR cDNA under the control of human hepatic apolipoprotein A2 promoter that targets the adeno-associated virus integration site AAVS1. We differentiated both FH-iPSCs and corrected FH-iPSCs (corr-FH-iPSCs) into hepatocytes to study statin-mediated regulation of genes involved in cholesterol metabolism. Upon HCV particle inoculation, viral replication and production were quantified in these cells. We showed that FH-iPSCs displayed the disease phenotype. Using homologous recombination mediated by the CRISPR/Cas9 system, FH-iPSCs were genetically corrected by the targeted integration of a correction cassette at the AAVS1 locus. Both FH-iPSCs and corr-FH-iPSCs were then differentiated into functional polarized hepatocytes using a stepwise differentiation approach (FH-iHeps and corr-FH-iHeps). The correct insertion and expression of the correction cassette resulted in restoration of LDLR expression and function (LDL-c uptake) in corr-FH-iHeps. We next demonstrated that pravastatin treatment increased the expression of genes involved in cholesterol metabolism in both cell models. Moreover, LDLR expression and function were also enhanced in corr-FH-iHeps after pravastatin treatment. Finally, we demonstrated that both FH-iHeps and corr-FH-iHeps were as permissive to viral infection as primary human hepatocytes but that virus production in FH-iHeps was significantly decreased compared to corr-FH-iHeps, suggesting a role of the LDLR in HCV morphogenesis. Our work provides the first LDLR-null FH cell model and its corrected counterpart to study the regulation of cholesterol metabolism and host determinants of HCV life cycle, and a platform to screen drugs for treating dyslipidemia and HCV infection.

Genomic integrity of human induced pluripotent stem cells: Reprogramming, differentiation and applications

Background/Objectives: Human pluripotent stem cells (hPSCs) and organoid technologies are transforming pharmaceutical research by providing models that more accurately reflect human physiology, genetic variability, and disease mechanisms. This review aims to assess how these systems improve the predictive power of preclinical drug development while addressing ethical concerns and supporting the advancement of precision medicine. Methods: We conducted a comprehensive review of the recent literature focusing on the biological principles, technological developments, and pharmaceutical applications of hPSC- and organoid-based systems. Particular attention was given to patient-derived models, integration of omics approaches, bioengineering advances, and artificial intelligence applications in drug screening workflows. Results: hPSC- and organoid-based platforms outperform traditional 2D cultures and animal models in replicating human-specific pathophysiology, enabling personalized drug testing and improving predictions of therapeutic efficacy and safety. These technologies also align with the ethical principles of the 3Rs (replacement, reduction, and refinement) by reducing reliance on animal experimentation. However, challenges persist, including standardization of protocols, batch-to-batch variability, and scalability. Promising solutions involve automation, high-throughput screening, and multi-omics integration, which collectively enhance reproducibility and translational relevance. Conclusions: Stem cell- and organoid-based systems offer a more human-relevant, ethical, and individualized approach to biomedical research. Despite current limitations, ongoing interdisciplinary innovations are expected to accelerate their clinical and industrial adoption. Collaborative efforts will be essential to standardize methodologies and fully realize the potential of these models in bridging preclinical and clinical drug development.

The use of synthetic messenger RNAs to generate human induced pluripotent stem cells (iPSCs) is particularly appealing for potential regenerative medicine applications, because it overcomes the common drawbacks of DNA-based or virus-based reprogramming strategies, including transgene integration in particular. We compared the genomic integrity of mRNA-derived iPSCs with that of retrovirus-derived iPSCs generated in strictly comparable conditions, by single-nucleotide polymorphism (SNP) and copy number variation (CNV) analyses. We showed that mRNA-derived iPSCs do not differ significantly from the parental fibroblasts in SNP analysis, whereas retrovirus-derived iPSCs do. We found that the number of CNVs seemed independent of the reprogramming method, instead appearing to be clone-dependent. Furthermore, differentiation studies indicated that mRNA-derived iPSCs differentiated efficiently into hepatoblasts and that these cells did not load additional CNVs during differentiation. The integration-free hepatoblasts that were generated constitute a new tool for the study of diseased hepatocytes derived from patients' iPSCs and their use in the context of stem cell-derived hepatocyte transplantation. Our findings also highlight the need to conduct careful studies on genome integrity for the selection of iPSC lines before using them for further applications.