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.

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.

Reliable storage and distribution are important for developing ready-to-use encapsulated cell products for transplantation and bioartificial liver applications. This study evaluated the cryopreservation of alginate-encapsulated HepaRG cells using a programmable controlled-rate freezing (CRF) protocol at −0.3 °C/min or conventional passive cooling at approximately −1 °C/min. Five media were tested: four commercial xeno-free formulations, including two DMSO-containing and two DMSO-free media, and a conventional formulation containing 90% fetal bovine serum and 10% DMSO. Following thawing and a 3-day recovery period, metabolic activity, albumin secretion, ammonia and lactate clearance, and phase I and phase II biotransformation activities were assessed. Cryopreservation reduced metabolic activity relative to non-frozen controls, but key hepatic functions remained detectable. The tested CRF protocol generally yielded equal or higher functional values than conventional freezing. Medium performance varied according to the endpoint assessed. Within CRF, CryoStor CS10 produced relatively high mean PrestoBlue and albumin secretion values, whereas Cellvation produced the highest mean CYP1A1/1A2 activity. These product-specific observations do not establish a general effect of DMSO. The findings support the short-term feasibility of the tested CRF protocol for this encapsulated HepaRG model but not broader clinical or drug-testing applications.