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.

Background: Human-induced pluripotent stem cell-derived hepatocytes (iHeps) have been shown to have considerable potential in liver diseases, toxicity, and pharmacological studies. However, there is a growing need to obtain iHeps that are truly similar to primary adult hepatocytes in terms of morphological features and functions. We generated such human iHeps, self-assembled as organoids (iHep-Orgs). Methods: iPSC-derived hepatoblasts were self-assembled into spheroids and differentiated into mature hepatocytes modulating final step of differentiation. Results: In about four weeks of culture, the albumin secretion levels and the complete disappearance of α-fetoprotein from iHep-Orgs suggested the acquisition of a greater degree of maturation than those previously reported. The expression of apical transporters and bile acid secretion evidenced the acquisition of complex hepatocyte polarity as well as the development of a functional and well-defined bile canalicular network confirmed by computational analysis. Activities recorded for CYP450, UGT1A1, and alcohol dehydrogenase, response to hormonal stimulation, and glucose metabolism were also remarkable. Finally, iHep-Orgs displayed a considerable ability to detoxify pathological concentrations of lactate and ammonia. Conclusions: With features similar to those of primary adult hepatocytes, the iHep-Orgs thus produced could be considered as a valuable tool for the development and optimization of preclinical and clinical applications.

Pluripotent stem cell-derived hepatocytes differentiated in monolayer culture are known to have more foetal than adult hepatocyte characteristics. If numerous studies tend to show that this immature phenotype might not necessarily be an obstacle to their use in transplantation, other applications such as drug screening, toxicological studies or bioartificial livers are reliant on hepatocyte functionality and require full differentiation of hepatocytes. New technologies have been used to improve the differentiation process in recent years, usually evaluated by measuring the albumin production and CYP450 activity. Here we used the complex production and most importantly the activity of the coagulation factor IX (FIX) produced by mature hepatocytes to assess the differentiation of Hemophilia B patient’s induced pluripotent stem cells (iPSCs) in both monolayer culture and organoids. Indeed, Hemophilia B (HB) is an X-linked monogenic disease due to an impaired activity of FIX synthesized by hepatocytes in the liver. We have developed an in vitro model of HB hepatocytes using iPSCs generated from fibroblasts of a severe HB patient. We used CRISPR/Cas9 technology to target the genomic insertion of an F9 mini-gene bearing the Padua mutation to enhance FIX activity. Non-corrected and corrected iPSCs were differentiated into hepatocytes under both 2D and 3D differentiation protocols and deciphered the production of active FIX in vitro. Functional FIX, whose post-translational modifications only occur in fully mature hepatocytes, was only produced in corrected iPSCs differentiated in organoids. Finally, we assessed the therapeutic efficacy of this approach in vivo using a mouse model of HB. Immunohistochemistry analyses indicated a good cell engraftment and the FIX activity detected in the plasma of transplanted animals confirmed rescue of the bleeding phenotype.

Liver cell therapy and in vitro models require functional human hepatocytes, the sources of which are considerably limited. Human induced pluripotent stem cells (hiPSCs) represent a promising and unlimited source of differentiated human hepatocytes. However, when obtained in two-dimensional (2D) cultures these hepatocytes are not fully mature and functional. As three-dimensional culture conditions offer advantageous strategies for differentiation, we describe here a combination of three-dimensional (3D) approaches enabling the successful differentiation of functional hepatocytes from hiPSCs by the encapsulation of hiPSC-derived hepatoblasts in alginate beads of preformed aggregates. The resulting encapsulated and differentiated hepatocytes (E-iHep-Orgs) displayed a high level of albumin synthesis associated with the disappearance of α-fetoprotein (AFP) synthesis, thus demonstrating that the E-iHep-Orgs had reached a high level of maturation, similar to that of adult hepatocytes. Gene expression analysis by RT-PCR and immunofluorescence confirmed this maturation. Further functional assessments demonstrated their enzymatic activities, including lactate and ammonia detoxification, as well as biotransformation activities of Phase I and Phase II enzymes. This study provides proof of concept regarding the benefits of combining three-dimensional techniques (guided aggregation and microencapsulation) with liver differentiation protocols as a robust approach to generate mature and functional hepatocytes that offer a permanent and unlimited source of hepatocytes. Based on these encouraging results, our combined conditions to produce mature hepatocytes from hiPSCs could be extended to liver tissue engineering and bioartificial liver (BAL) applications at the human scale for which large biomasses are mandatory.

Drug-induced liver injury, also known as drug-induced hepatotoxicity (DILI), is a major cause of medicine withdrawal (prescription or over-the-counter) from the market