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Background & Aim: Shortage of donor hepatocytes limits hepatocyte transplantation for clinical application. Induced hepatic stem cells (iHepSCs) have capacities of self-renewal and bipotential differentiations. Here, we investigated whether iHepSCs could be extensively expanded, and whether they could differentiate into sufficient functional hepatocytes as donors for transplantation therapy after their extensive expansions., Methods: Murine extensively expanded iHepSCs (50-55 passages) were induced to differentiate into iHepSC-Heps under a chemically defined condition. iHepSC-Heps were proved for carrying morphological hepatocyte characters and hepatocytic functions including low-density lipoprotein uptake, glycogen storage, CLF secretion, ICG uptake and release, Alb secretion, urea synthesis and metabolism-relative gene expressions respectively. Next, both iHepSCs and iHepSC-Heps were transplanted into Fah-/- mice respectively. Both liver repopulation and alleviation of liver function were compared between two transplantation groups., Results: Murine iHepSCs still maintained the capacities of self-renewal and bipotential differentiations after extensive expansion. The efficiency for the functional hepatocyte differentiation from extensively expanded iHepSCs reached to 72.64%. Transplantations of both extensively expanded iHepSCs and iHepSC-Heps resulted in liver engraftment in Fah-/- mice. Survival rate of Fah-/- mice recipients and level of liver repopulation were 50% and 20.32 ± 4.58% respectively in iHepSC-Heps group, while 33% and 10.4 ± 4.3% in iHepSCs group., Conclusions: Extensively expanded iHepSCs can efficiently differentiate into hepatocytes in chemical defined medium. Transplantation of iHepSC-Heps was more effective and more efficient than transplantation of iHepSCs in Fah-/- mice. Our results suggested an innovative system to obtain sufficient hepatocytes through hepatic differentiation of iHepSCs generated by lineage reprogramming., (© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Functional human hepatocytes xenografted into the liver of mice can be used as a model system to study pharmacokinetics, infection of hepatitis viruses, and the efficacy of hepatitis vaccines. Significant levels of liver xeno-repopulation have been reported in Fah (-/-) Rag2 (-/-) Il2rg (-/-) mice. However, A new model, termed Fah (-/-) Nod/Scid mice, which combines the advantages of liver repopulation in Fah (-/-) mice with the ease of xenotransplantation in Nod/Scid mice was obtained by gradual cross-breeding. Fah (-/-) Nod/Scid mice were easily maintained in breeding colonies and in adult animal care facilities. FK506 treatment combined with gradual withdrawal of NTBC before cell transplantation ensured that Fah (-/-) Nod/Scid mice were susceptible to liver xeno-repopulation by human hepatocytes; the proportion of engrafted human hepatocytes reached 33.6%. The function of the expanded human hepatocytes within the chimeric liver was confirmed by weight curve analysis, the expression of characteristic proteins, and the biochemical analysis of liver function. These results show that Fah (-/-) Nod/Scid mice are an ideal humanized liver mouse model with many useful applications.

Functional human hepatocytes xeno-engrafted in mouse liver can be used as a model system to study hepatitis virus infection and vaccine efficacy. Significant liver xeno-repopulation has been reported in two kinds of genetically modified mice that have both immune deficiency and liver injury-induced donor hepatocyte selection: the uPA/SCID mice and Fah(-/-) Rag2(-/-)Il2rg(-/-) mice. The lack of hardy breeding and the overly elaborated technique in these two models may hinder the potential future application of these models to hepatitis virus infection and vaccination studies. Improving the transplantation protocol for liver xeno-repopulation from human hepatocytes will increase the model efficiency and application. In this study, we successfully apply immunosuppressive drug treatments of anti-asialo GM1 and FK506 in Fah(-/-)Rag2(-/-) mice, resulting in significant liver xeno-repopulation from human hepatocytes and human fetal liver cells. This methodology decreases the risk of animal mortality during breeding and surgery. When infected with hepatitis B virus (HBV) sera, Fah(-/-)Rag2(-/-) mice with liver xeno-repopulation from human hepatocytes accumulate significant levels of HBV DNA and HBV proteins. Our new protocol for humanized liver could be applied in the study of human hepatitis virus infection in vivo, as well as the pharmacokinetics and efficacy of potential vaccines.

The liver regenerates by progenitor cells when it is damaged in chronic liver diseases and extensive damage. The progenitor cells, also termed "oval cells" according to their morphological traits, can differentiate into hepatocytes and bile duct cells in vivo. To better understand the transcriptional pattern that accompanies the hepatic differentiation of oval cells, we applied cDNA microarray to analyze the oval cell-derived liver epithelial progenitor cells (LEPCs) during in vitro induced differentiation. Upon exposure to sodium butyrate, a histone deacetylase inhibitor, cultured LEPCs differentiate and express functional hepatocyte markers albumin, tryptophan 2, 3-dioxygenase and alcohol dehydrogenase. For expression profiling, cells were harvested at 6 h, 12 h, 1 d, 3 d and 7 d after exposure to sodium butyrate. After analyzing the microarray data by SOM clustering, total of 796 differentially regulated genes were grouped into 48 clusters. Consistent with the phenotype change of LEPCs after sodium butyrate treatment, many hepatocyte functional genes are revealed by analyzing the clusters containing genes up-regulated through all the time points. The clusters, containing down-regulated genes immediately after the induction, are also analyzed. The microarray data was validated by analyzing the expression of selected genes by quantitative real-time PCR. A set of genes expressed synergistically in these clusters may play a central role during the process of differentiation. Sodium butyrate decreases cyclin B1 and Cdk4 expression, which would be associated with LEPCs growth arrest shortly after treatment. Bmi1, a polycomb group protein, is also down-regulated immediately after treatment and remains at a low level during the induction. These findings highlight the key molecular mechanisms by which sodium butyrate, mediates its effects on cell growth arrest and induction of differentiation. In conclusion, our data reflect a global view of gene expression during hepatic differentiation of LEPCs induced by sodium butyrate.