Development of human iPSC-derived quiescent hepatic stellate cell-like cells for drug discovery and in vitro disease modeling.

Hepatic stellate cells (HSCs) play a central role in the progression of liver fibrosis by producing extracellular matrices. The development of drugs to suppress liver fibrosis has been hampered by the lack of human quiescent HSCs (qHSCs) and an appropriate in vitro model that faithfully recapitulates HSC activation. In the present study, we developed a culture system to generate qHSC-like cells from human-induced pluripotent stem cells (hiPSCs) that can be converted into activated HSCs in culture. To monitor the activation process, a red fluorescent protein (RFP) gene was inserted in hiPSCs downstream of the activation marker gene actin alpha 2 smooth muscle (ACTA2). Using qHSC-like cells derived from RFP reporter iPSCs, we screened a repurposing chemical library and identified therapeutic candidates that prevent liver fibrosis. Hence, hiPSC-derived qHSC-like cells will be a useful tool to study the mechanism of HSC activation and to identify therapeutic agents.

Wnt5a signaling mediates biliary differentiation of fetal hepatic stem/progenitor cells in mice.

UNLABELLED: The molecular mechanisms regulating differentiation of fetal hepatic stem/progenitor cells, called hepatoblasts, which play pivotal roles in liver development, remain obscure. Wnt signaling pathways regulate the development and differentiation of stem cells in various organs. Although a ß-catenin-independent noncanonical Wnt pathway is essential for cell adhesion and polarity, the physiological functions of noncanonical Wnt pathways in liver development are unknown. Here we describe a functional role for Wnt5a, a noncanonical Wnt ligand, in the differentiation of mouse hepatoblasts. Wnt5a was expressed in mesenchymal cells and other cells of wild-type (WT) midgestational fetal liver. We analyzed fetal liver phenotypes in Wnt5a-deficient mice using a combination of histological and molecular techniques. Expression levels of Sox9 and the number of hepatocyte nuclear factor (HNF)1ß(+) HNF4α(-) biliary precursor cells were significantly higher in Wnt5a-deficient liver relative to WT liver. In Wnt5a-deficient fetal liver, in vivo formation of primitive bile ductal structures was significantly enhanced relative to WT littermates. We also investigated the function of Wnt5a protein and downstream signaling molecules using a three-dimensional culture system that included primary hepatoblasts or a hepatic progenitor cell line. In vitro differentiation assays showed that Wnt5a retarded the formation of bile duct-like structures in hepatoblasts, leading instead to hepatic maturation of such cells. Whereas Wnt5a signaling increased steady-state levels of phosphorylated calcium/calmodulin-dependent protein kinase II (CaMKII) in fetal liver, inhibition of CaMKII activity resulted in the formation of significantly more and larger-sized bile duct-like structures in vitro compared with those in vehicle-supplemented controls. CONCLUSION: Wnt5a-mediated signaling in fetal hepatic stem/progenitor cells suppresses biliary differentiation. These findings also suggest that activation of CaMKII by Wnt5a signaling suppresses biliary differentiation. (HEPATOLOGY 2013;).

Isolation of hepatoblasts based on the expression of Dlk/Pref-1.

Hepatoblasts are common progenitors for hepatocytes and biliary epithelial cells, although their nature remains largely unknown. In order to isolate and to characterize hepatoblasts, we searched for cell surface antigens expressed in mouse fetal hepatic cells by the signal sequence trap method and found that Dlk, also known as Pref-1, was strongly expressed in fetal liver. Immunohistochemical as well as northern analysis indicated that Dlk was highly expressed in the E10.5 liver bud. The strong expression continued until the E16.5 stage and was significantly downregulated thereafter. Using a monoclonal antibody against Dlk, we isolated Dlk+ cells either by a fluorescence-activated cell sorter or by an automatic magnetic cell sorter. Dlk+ cells isolated from fetal livers expressed albumin and formed colonies when cultured at low density with HGF and EGF for 5 days. Over 60% of colonies derived from E14.5 Dlk+ cells contained both albumin+ and cytokeratin 19+ cells, indicating that a majority of colony-forming Dlk+ cells are able to differentiate into both hepatocyte and biliary epithelial cell lineages. In addition, numerous microvilli were observed by electronmicroscopic analysis in most of those cultured cells, also indicating differentiation of Dlk+ cells under this condition. Furthermore, 7% of the colony-forming Dlk+ cells were not only bipotential but also highly proliferative, forming a large colony containing more than 100 cells during 5 days of culture. By transplantation of Dlk+ cells into the spleen, donor-derived hepatocytes were found in the recipient liver, indicating that Dlk+ cells differentiated into hepatocytes in vivo. These results indicate that Dlk+ cells are hepatoblasts and that Dlk is a useful marker to enrich highly proliferative hepatoblasts from fetal liver.