Fibrosis is a part of the wound-healing response to tissue damage and characterized by excessive accumulation of mainly type I collagen-containing extracellular matrices (ECMs). Transforming growth factor beta (TGF-β) is a profibrogenic master cytokine responsible for promoting differentiation of tissue-resident fibroblasts into myofibroblasts, upregulation of ECM production, and downregulation of ECM degradation. The formation of ECM is an essential response in wound healing. Fibronectin is an ECM glycoprotein substantially expressed during tissue repair. Based on in vitro findings, it has been widely accepted that collagen network organization was exclusively fibronectin matrix dependent. Unexpectedly, our fibronectin conditional knockout mouse models have demonstrated a fibronectin-independent mechanism of collagen fibril formation following injury and identified TGF-β signaling and type V collagen as essential elements for collagen fibrillogenesis. Interestingly, the targeting of the TGF-β signaling alone, as proposed in some recent antifibrotic therapies of chronic fibrotic diseases, is not sufficient to completely prevent liver fibrosis. In this chapter, we focus on the present knowledge of the mechanisms of the collagen network organization following tissue/organ damage and pathological processes of chronic fibrotic diseases.

Automation of cell culture would facilitate stable cell expansion with consistent quality. In the present study, feasibility of an automated closed-cell culture system “P 4C S” for an embryoid body- (EB-) explant outgrowth culture was investigated as a model case for explant culture. After placing the induced pluripotent stem cell- (iPSC-) derived EBs into the system, the EBs successfully adhered to the culture surface and the cell outgrowth was clearly observed surrounding the adherent EBs. After confirming the outgrowth, we carried out subculture manipulation, in which the detached cells were simply dispersed by shaking the culture flask, leading to uniform cell distribution. This enabled continuous stable cell expansion, resulting in a cell yield of 3.1 × 107. There was no evidence of bacterial contamination throughout the cell culture experiments. We herewith developed the automated cultivation platform for EB-explant outgrowth cells.

No abstract

Kynurenine (Kyn), a metabolite of tryptophan (Trp), is a key regulator of mammal immune responses such as cancer immune tolerance. Indoleamine-2,3-dioxygenase (IDO) and tryptophan-2,3-dioxygenase (TDO) are main enzymes regulating the first and rate-limiting step of the Kyn pathway. To identify new small molecule inhibitors of TDO, we selected A172 glioblastoma cell line constitutively expressed TDO. Characterization of this cell line using kinase inhibitor library resulted in identification of MEK/ERK pathway-dependent TDO expression. After knowing the properties for TDO expression, we further proceeded to screen chemical library for TDO inhibitors. We previously determined that S-benzylisothiourea derivatives are enzymatic inhibitors of indoleamine 2,3-dioxygenase 1 (IDO1) and suggested that the isothiourea moiety could be an important pharmacophore for binding to heme. Based on this premise, we screened an in-house library composed of various isothiourea derivatives and identified a bisisothiourea derivative, PVZB3001, as an inhibitor of TDO. Interestingly, PVZB3001 also inhibited the enzymatic activity of IDO1 in both cell-based and cell-free assays but did not inhibit other heme enzymes. Molecular docking studies suggested the importance of isothiourea moieties at the ortho position of the phenyl ring for the inhibition of catalytic activity. PVZB3001 showed competitive inhibition against TDO, and this was supported by the docking simulation. PVZB3001 recovered natural killer (NK) cell viability and functions by inhibiting Kyn accumulation in conditioned medium of both IDO1- and TDO-expressing cells. Furthermore, oral administration of IDO1-overexpressing tumor-bearing mice with PVZB3001 significantly inhibited tumor growth. Thus, we identified a novel selective dual inhibitor of IDO1 and TDO using the Kyn production assay with a glioblastoma cell line. This inhibitor could be a useful pharmacological tool for modulating the Kyn pathway in a variety of experimental systems.

Many drugs have the potential to induce the expression of drug-metabolizing enzymes, particularly cytochrome P450 3A4 (CYP3A4). Hepatocytes are often employed to evaluate drug-mediated CYP3A4 induction, but the variation between different cell lots is an issue that needs to be solved. Only a limited number of immortalized hepatocyte cell lines have been reported to date. In this study, we describe the successful generation of hepatocytes from disease-specific induced pluripotent stem cells (iPSCs) derived from a patient with fulminant hepatitis (FH-iPSCs). To examine the CYP3A4 induction potential, FH-iPSCs were induced into hepatocytes. Drug-mediated induction testing revealed that HepaKI exhibited a 57.2-fold increase in CYP3A4 after exposure to rifampicin, relative to control cells. These results suggest that FH-iPSCs are a preferred cell source for in vitro CYP3A4 induction assays.