In vitro characterization of 3D culture-based differentiation of human liver stem cells

A shortage of functional hepatocytes hampers drug safety testing and therapeutic applications because mature hepatocytes cannot be expanded and tend to lose functionality in vitro. Previous studies indicated that human liver stem cells (HLSCs) can differentiate into hepatocyte-like cells in an in vitro rotary cell culture system (RCCS), in the presence of different combinations of cytokines and molecules known to play a role in hepatocyte maturation. To characterize the hepatocyte-like cells obtained after HLSC differentiation, we analyzed the expression of stem and hepatic markers by qRTPCR and immunofluorescence and developed an updated Indocyanine green in vitro assay to measure hepatocyte functionality. Our results demonstrated that HLSCs, after one day of differentiation significantly upregulated the expression of hepatic genes, such as CYP 450, urea cycle enzymes and uptake transporters exclusively expressed on the sinusoidal side of mature hepatocytes. In addition, during differentiation, it has been observed a downregulation of stem cell markers, such as Klf4. Further analysis showed that the differentiated cells were able to release urea and FVIII into the supernatant, as early as in the first 24 hours which accumulate over time. In conclusion, the results suggested that the three-dimensional RCCS may promote the rapid hepatic differentiation of HLSCs. Despite the limitations of this rotary culture system, it possesses unique advantages and in addition may provide a potential strategy to characterize HLSCs GMP batches for clinical application.