Human pluripotent stem cell-derived liver organoids (HLOs) have recently become a promising alternative for liver regenerative therapy. To realize this application, a large amount of human-induced pluripotent stem cells (hiPSCs) derived-liver cells are required for partial liver replacement during transplantation. This method requires stepwise induction using costly growth factors to direct the hiPSCs into the hepatic lineage. Therefore, we developed a simple dialysis-based medium conditioning that fully utilized growth factors accumulation to improve hepatic differentiation of hiPSCs at a high cell density. The results demonstrated that the dialysis culture system could accumulate the four essential growth factors required in each differentiation stage: activin A, bone morphogenetic protein 4 (BMP4), hepatocyte growth factor (HGF), and oncostatin M (OSM). As a result, this low lactate culture environment allowed high-density bipotential hepatic differentiation of up to 4.5 × 107 cells/mL of human liver organoids (HLOs), consisting of hiPSC derived-hepatocyte like cells (HLCs) and cholangiocyte like-cells (CLCs). The differentiated HLOs presented a better or comparable hepatic marker and hepatobiliary physiology to the one that differentiated in suspension culture with routine daily medium replacement at a lower cell density. This simple miniaturized dialysis culture system demonstrated the feasibility of cost-effective high-density hepatic differentiation with minimum growth factor usage.

Oxygen, as an external environmental factor, plays a role in the early differentiation of human stem cells, such as induced pluripotent stem cells (hiPSCs). However, the effect of oxygen concentration on the early-stage differentiation of hiPSC is not fully understood, especially in 3D aggregate cultures. In this study, we cultivated the 3D aggregation of hiPSCs on oxygen-permeable microwells under different oxygen concentrations ranging from 2.5 to 20% and found that the aggregates became larger, corresponding to the increase in oxygen level. In a low oxygen environment, the glycolytic pathway was more profound, and the differentiation markers of the three germ layers were upregulated, suggesting that the oxygen concentration can function as a regulator of differentiation during the early stage of development. In conclusion, culturing stem cells on oxygen-permeable microwells may serve as a platform to investigate the effect of oxygen concentration on diverse cell fate decisions during development.

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The human pluripotent stem cells-derived liver organoid (HLOs) has recently become a promising alternative source for liver regenerative therapy. To realize this application, a large number of the hiPSCs derived-liver cells are required for partial liver replacement in transplantation. This method requires a stepwise induction by using costly growth factors to direct the hiPSCs into the hepatic lineage. Therefore, we developed a simple dialysis culture system to fully utilize the growth factors accumulation with continuous medium refinement. The results showed that the dialysis culture system was able to accumulate the four essential growth factors required in each differentiation stage, such as Activin A, BMP4, HGF, and OSM. As a result, this culture system allowed a very high-density bipotential hepatic differentiation in approximately 4.5×107 cells/mL of human liver organoids (HLOs), mainly consisting of hiPSCs derived-hepatocytes (HLCs) and cholangiocytes (CLCs). Moreover, the differentiated liver organoids showed a better or comparable hepatic marker and physiological activity with the organoids differentiated in suspension culture with conventional daily medium replacement in a lower cell density. This simple miniaturized dialysis culture system demonstrated the feasibility of cost-effective hepatic differentiation in high density.