Hiroki Saito

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.

Hepatocyte proliferation and tissue remodeling is impaired after liver injury in oncostatin M receptor knockout mice

Oncostatin M (OSM) is a member of the IL‐6 family of cytokines. Mice deficient in the OSM receptor (OSMR‐/‐) showed impaired liver regeneration with persistent parenchymal necrosis after carbon tetrachloride (CCl4) exposure. The recovery of liver mass from partial hepatectomy was also significantly delayed in OSMR‐/‐ mice. In contrast to wildtype mice, CCl4 administration only marginally induced expression of tissue inhibitor of metalloproteinase (TIMP)‐1 and TIMP‐2 genes in OSMR‐/‐ mice, correlating with the increased gelatinase activity of matrix metalloproteinase (MMP)‐9 and matrix degradation in injured livers. The activation of STAT3 and expression of immediate early genes and cyclins were decreased in OSMR‐/‐ liver, indicating that OSM signaling is required for hepatocyte proliferation and tissue remodeling during liver regeneration. We also found that CCl4 administration in IL‐6‐/‐ mice failed to induce OSM expression and that OSM administration in IL‐6‐/‐ mice after CCl4 injection induced the expression of cyclin D1 and proliferating cell nuclear antigen, suggesting that OSM is a key mediator of IL‐6 in liver regeneration. Consistent with these results, administration of OSM ameliorated liver injury in wildtype mice by preventing hepatocyte apoptosis as well as tissue destruction. In conclusion, OSM and its signaling pathway may provide a useful therapeutic target for liver regeneration. (HEPATOLOGY 2004;39:635–644.)

Long-term culture of hepatic progenitors derived from mouse Dlk+ hepatoblasts

We previously demonstrated that hepatoblasts can be isolated from mouse fetal liver based on the expression of delta-like leucine zipper kinase (Dlk), also known as Pref-1. Each Dlk+ hepatoblast forms a colony containing both albumin+ hepatocytes and cytokeratin 19+ (CK19) cholangiocytic cells on either type IV collagen or laminin. Here we show that extracellular matrices (ECMs) significantly affect the growth of Dlk+ cells. Dlk+ cells vigorously proliferated on type IV collagen-coated dishes in the presence of EGF and HGF during the first 5 days, but their proliferative capability declined thereafter. Dlk+ cells also proliferated on laminin-coated plates and some colonies continued to expand even beyond one month after plating. These hepatic progenitor cells proliferating on laminin (HPPL) efficiently proliferated even after replating. Moreover, they were induced to differentiate into hepatocytes and cholangiocytes by overlaying Engelbreth-Holm-Swarm sarcoma (EHS) gel and by embedding in type I collagen gel, respectively. HPPL acquired the metabolic functions of accumulating polysaccharides and detoxifying ammonium ions after hepatic differentiation. Surprisingly, HPPL expressed pancreatic genes such as Pdx1 when dexamethasone was depleted from the culture medium. Therefore, the long-term culture of hepatoblasts on laminin produces multi-potential hepatic progenitors, which possess a strong proliferative capability, differentiate into both hepatocytes and cholangiocytes, and potentially give rise to pancreatic cells.

Two-tone pseudo coloring: compact visualization for one-dimensional data

A new pseudo coloring technique for large scale one-dimensional datasets is proposed. For visualization of a large scale dataset, user interaction is indispensable for selecting focus areas in the dataset. However, excessive switching of the visualized image makes it difficult for the user to recognize overview/ detail and detail/ detail relationships. The goal of this research is to develop techniques for visualizing details as precisely as possible in overview display. In this paper, visualization of a one-dimensional but very large dataset is considered. The proposed method is based on pseudo coloring, however, each scalar value corresponds to two discrete colors. By painting with two colors at each value, users can read out the value precisely. This method has many advantages: it requires little image space for visualization; both the overview and details of the dataset are visible in one image without distortion; and implementation is very simple. Several application examples, such as meteorological observation data and train convenience evaluation data, show the effectiveness of the method.

Generation of Glucose-Responsive Functional Islets with a Three-Dimensional Structure from Mouse Fetal Pancreatic Cells and iPS Cells In Vitro

Islets of Langerhans are a pancreatic endocrine compartment consisting of insulin-producing β cells together with several other hormone-producing cells. While some insulin-producing cells or immature pancreatic cells have been generated in vitro from ES and iPS cells, islets with proper functions and a three-dimensional (3D) structure have never been successfully produced. To test whether islets can be formed in vitro, we first examined the potential of mouse fetal pancreatic cells. We found that E16.5 pancreatic cells, just before forming islets, were able to develop cell aggregates consisting of β cells surrounded by glucagon-producing α cells, a structure similar to murine adult islets. Moreover, the transplantation of these cells improved blood glucose levels in hyperglycemic mice. These results indicate that functional islets are formed in vitro from fetal pancreatic cells at a specific developmental stage. By adopting these culture conditions to the differentiation of mouse iPS cells, we developed a two-step system to generate islets, i.e. immature pancreatic cells were first produced from iPS cells, and then transferred to culture conditions that allowed the formation of islets from fetal pancreatic cells. The islets exhibited distinct 3D structural features similar to adult pancreatic islets and secreted insulin in response to glucose concentrations. Transplantation of the islets improved blood glucose levels in hyperglycemic mice. In conclusion, the two-step culture system allows the generation of functional islets with a 3D structure from iPS cells.

Bose-Einstein Condensation with Attractive Interaction Fate of a False Vacuum

Among the species that have Bose-condensed, lithium 7 and rubidium 85 form unique condensates in that atoms interact attractively and that the energy barrier that stabilizes the gaseous Bose-Einstein condensates originates from the zero-point motion of atoms. This paper reviews some unique features of these new states of matter that are distinguished from those of other species with repulsive interactions. These include the density instability, macroscopic quantum tunneling, intermittent implosion, shellstructure formation, and partial quantization of circulation.