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In the liver, the bile canaliculi of hepatocytes are connected to intrahepatic bile ducts lined with cholangiocytes, which remove cytotoxic bile from the liver tissue. Although liver organoids have been reported, it is not clear whether the functional connection between hepatocytes and cholangiocytes is recapitulated in those organoids. Here, we report the generation of a hepatobiliary tubular organoid (HBTO) using mouse hepatocyte progenitors and cholangiocytes. Hepatocytes form the bile canalicular network and secrete metabolites into the canaliculi, which are then transported into the biliary tubular structure. Hepatocytes in HBTO acquire and maintain metabolic functions including albumin secretion and cytochrome P450 activities, over the long term. In this study, we establish functional liver tissue incorporating a bile drainage system ex vivo. HBTO enable us to reproduce the transport of hepatocyte metabolites in liver tissue, and to investigate the way in which the two types of epithelial cells establish functional connections. Combining mouse hepatocyte progenitors and cholangiocytes ex vivo, the authors form an organoid that can drain bile ex vivo and transport metabolites, as in the liver.

Nonalcoholic steatohepatitis (NASH) is a metabolic liver disease that progresses from simple steatosis to the disease state of inflammation and fibrosis. Previous studies suggest that apoptosis and necroptosis may contribute to the pathogenesis of NASH, based on several murine models. However, the mechanisms underlying the transition of simple steatosis to steatohepatitis remain unclear, because it is difficult to identify when and where such cell deaths begin to occur in the pathophysiological process of NASH. In the present study, our aim is to investigate which type of cell death plays a role as the trigger for initiating inflammation in fatty liver. By establishing a simple method of discriminating between apoptosis and necrosis in the liver, we found that necrosis occurred prior to apoptosis at the onset of steatohepatitis in the choline-deficient, ethionine-supplemented (CDE) diet model. To further investigate what type of necrosis is involved in the initial necrotic cell death, we examined the effect of necroptosis and ferroptosis inhibition by administering inhibitors to wild-type mice in the CDE diet model. In addition, necroptosis was evaluated using mixed lineage kinase domain-like protein (MLKL) knockout mice, which is lacking in a terminal executor of necroptosis. Consequently, necroptosis inhibition failed to block the onset of necrotic cell death, while ferroptosis inhibition protected hepatocytes from necrotic death almost completely, and suppressed the subsequent infiltration of immune cells and inflammatory reaction. Furthermore, the amount of oxidized phosphatidylethanolamine, which is involved in ferroptosis pathway, was increased in the liver sample of the CDE diet-fed mice. These findings suggest that hepatic ferroptosis plays an important role as the trigger for initiating inflammation in steatohepatitis and may be a therapeutic target for preventing the onset of steatohepatitis.

The liver is a unique organ with an extraordinary capacity to regenerate upon various injuries. In acute and transient liver injury by insults such as chemical hepatotoxins, the liver in rodents returns to the original architecture by proliferation and remodeling of the remaining cells within a week. In contrast, chronic liver inflammation due to various etiologies, e.g., virus infection and metabolic and immune disorders, results in liver fibrosis, often leading to cirrhosis and carcinogenesis. In both acute and chronic inflammation, a variety of immune and non-immune cells in the liver is involved in the processes resulting in either regeneration or fibrosis. In addition, chronic hepatitis often accompanies proliferation of atypical biliary cells, also known as liver progenitor cells or oval cells. Although the origin of liver progenitor cells and its contribution to hepatic repair is still under intense debate, recent studies have revealed a regulatory role for immune cells in progenitor proliferation and differentiation. In this review, we summarize recent studies on liver regeneration and fibrosis in the viewpoint of inflammation.

Dynamic remodeling of the intrahepatic biliary epithelial tissue plays key roles in liver regeneration, yet the cellular basis for this process remains unclear. We took an unbiased approach based on in vivo clonal labeling and tracking of biliary epithelial cells in the three-dimensional landscape, in combination with mathematical simulation, to understand their mode of proliferation in a mouse liver injury model where the nascent biliary structure formed in a tissue-intrinsic manner. An apparent heterogeneity among biliary epithelial cells was observed: whereas most of the responders that entered the cell cycle upon injury exhibited a limited and tapering growth potential, a select population continued to proliferate, making a major contribution in sustaining the biliary expansion. Our study has highlighted a unique mode of epithelial tissue dynamics, which depends not on a hierarchical system driven by fixated stem cells, but rather, on a stochastically maintained progenitor population with persistent proliferative activity. Cell proliferation – the process by which cells multiply – plays an important role in many biological processes, including tissue growth, maintenance and remodeling. In these processes, the way cells proliferate is reportedly related to their roles in the tissue and the structures that they form. The biliary tree, a piping system that exists to drain the bile produced in the liver, forms a complex, tree-like, tubular structure. The biliary tree is essential for healthy livers to work well, and has been known to grow and change its structure quite dynamically during an injury or while the liver regenerates. However, it was not clear how biliary tree cells behave as the biliary tree grows and remodels itself. Does each cell behave in the same way? And how does cell growth relate to changes in the structure of the biliary tree? Kamimoto et al. have now developed new methods to observe detailed three-dimensional tissue structures and to trace the behavior of single cells. Using these techniques to study a mouse model whose liver was injured by toxic chemicals revealed the behavior of biliary cells as they responded to the injury. None of the biliary cells proliferated uniformly, and there were some peculiar cells that proliferated quite vigorously compared to the others. Kamimoto et al. then made a mathematical model that could explain cell behavior and tissue remodeling at different scales. This showed that the activity of those peculiar, rapidly proliferating cells was maintained by chance as the biliary tree expanded. These findings help us understand how the biliary tissue grows and the liver regenerates. They may also provide us with a clue to understanding the nature of the behavior of living things, which is sometimes seemingly ordered and robust, and sometimes unpredictable and mysterious. It remains to be seen whether the new model can be applied to other types of tissues or in other species. Further work is also needed to investigate which genes and proteins are involved in controlling the behavior of cells in the growing biliary tissue.

Chronic liver injury induces fibrosis that often proceeds to cirrhosis and hepatocellular carcinoma, indicating that prevention and/or resolution of fibrosis is a promising therapeutic target. Hepatic stellate cells (HSCs) are the major driver of fibrosis by expressing extracellular matrices (ECM). HSCs, in the normal liver, are quiescent and activated by liver injury to become myofibroblasts that proliferate and produce ECM. It has been shown that activated HSCs (aHSCs) become a \"quiescent-like\" state by removal of liver insults. Therefore, deactivation agents can be a therapeutic drug for advanced liver fibrosis. Using aHSCs prepared from human induced pluripotent stem cells, we found that aHSCs were reverted to a quiescent-like state by a combination of chemical compounds that either inhibit or activate a signaling pathway, Lanifibranor, SB431542, Dorsomorphin, retinoic acid, palmitic acid and Y27632, in vitro. Based on these results, we established a high throughput system to screen agents that induce deactivation and demonstrate that a single chemical compound can induce deactivation.

Liver fibrosis results from chronic liver injury triggered by factors such as viral infection, excess alcohol intake, and lipid accumulation. However, the mechanisms underlying liver fibrosis are not fully understood. Here, we demonstrate that the expression of fibroblast growth factor 18 ( Fgf18 ) is elevated in mouse livers following the induction of chronic liver fibrosis models. Deletion of Fgf18 in hepatocytes attenuates liver fibrosis; conversely, overexpression of Fgf18 promotes liver fibrosis. Single-cell RNA sequencing reveals that overexpression of Fgf18 in hepatocytes results in an increase in the number of Lrat + hepatic stellate cells (HSCs), thereby inducing fibrosis. Mechanistically, FGF18 stimulates the proliferation of HSCs by inducing the expression of Ccnd1 . Moreover, the expression of FGF18 is correlated with the expression of profibrotic genes, such as COL1A1 and ACTA2 , in human liver biopsy samples. Thus, FGF18 promotes liver fibrosis and could serve as a therapeutic target to treat liver fibrosis. Fibroblast growth factor (FGF)18 plays pleiotropic roles, including bone development and carcinogenesis, however, its precise role in liver fibrosis remains incompletely understood. Here, the authors show that FGF18 promotes liver fibrosis by stimulating hepatic stellate cell proliferation, without concomitant upregulation of profibrotic genes.

Hepatocytes derived from human iPSCs are useful to study hepatitis B virus (HBV) infection, however infection efficiency is rather poor. In order to improve the efficiency of HBV infection to iPSC-derived hepatocytes, we set a co-culture of hepatocytes with liver non-parenchymal cells and found that liver sinusoidal endothelial cells (LSECs) enhanced HBV infection by secreting epidermal growth factor (EGF). While EGF receptor (EGFR) is known as a co-receptor for HBV, we found that EGF enhanced HBV infection at a low dose of EGF, whereas EGF at a high dose suppressed HBV infection. EGFR is internalized by clathrin-mediated endocytosis (CME) and clathrin-independent endocytosis (CIE) pathways depending on the dose of EGF. At a high dose of EGF, the endocytosed EGFR via CIE is degraded in the lysosome. This study is the first to provide evidence that HBV is endocytosed via CME and CIE pathways at a low and high dose of EGF, respectively. In conclusion, we developed an in vitro system of HBV infection using iPSC-derived liver cells, and show that EGF secreted from LSECs modulates HBV infection in a dose dependent manner.

Mesenchymal stromal cells (MSCs) are used for ameliorating liver fibrosis and aiding liver regeneration after cirrhosis; Here, we analyzed the therapeutic potential of small extracellular vesicles (sEVs) derived from interferon-γ (IFN-γ) pre-conditioned MSCs (γ-sEVs). γ-sEVs effectively induced anti-inflammatory macrophages with high motility and phagocytic abilities in vitro, while not preventing hepatic stellate cell (HSC; the major source of collagen fiber) activation in vitro. The proteome analysis of MSC-derived sEVs revealed anti-inflammatory macrophage inducible proteins (e.g., annexin-A1, lactotransferrin, and aminopeptidase N) upon IFN-γ stimulation. Furthermore, by enabling CX3CR1+ macrophage accumulation in the damaged area, γ-sEVs ameliorated inflammation and fibrosis in the cirrhosis mouse model more effectively than sEVs. Single cell RNA-Seq analysis revealed diverse effects, such as induction of anti-inflammatory macrophages and regulatory T cells, in the cirrhotic liver after γ-sEV administration. Overall, IFN-γ pre-conditioning altered sEVs resulted in efficient tissue repair indicating a new therapeutic strategy.

The bone marrow (BM) is an essential organ for hematopoiesis in adult, in which proliferation and differentiation of hematopoietic stem/progenitor cells (HSPC) is orchestrated by various stromal cells. Alterations of BM hematopoietic environment lead to various hematopoietic disorders as exemplified by the linking of fatty marrow with increased adipogenesis to anemia or pancytopenia. Therefore, the composition of mesenchymal stromal cell (MSC)-derived cells in the BM could be crucial for proper hematopoiesis, but the mechanisms underlying the MSC differentiation for hematopoiesis remain poorly understood. In this study, we show that Oncostatin M (OSM) knock out mice exhibited pancytopenia advancing fatty marrow with age. OSM strongly inhibited adipogenesis from BM MSC in vitro, whereas it enhanced their osteogenesis but suppressed the terminal differentiation. Intriguingly, OSM allowed the MSC-derived cells to support the ex vivo expansion of HSPC effectively as feeder cells. Furthermore, the administration of OSM in lethally irradiated wild-type mice blocked fatty marrow and enhanced the recovery of HSPC number in the BM and peripheral blood cells after engraftment of HSPC. Collectively, OSM plays multiple critical roles in the maintenance and development of the hematopoietic microenvironment in the BM at a steady state as well as after injury.

Generation of pancreatic β cells from pluripotent stem cells is a key technology to develop cell therapy for insulin-dependent diabetes and considerable efforts have been made to produce β cells. However, due to multiple and lengthy differentiation steps, production of β cells is often unstable. It is also desirable to eliminate undifferentiated cells to avoid potential risks of tumorigenesis. To isolate β cell precursors from late stage pancreatic endocrine progenitor (EP) cells derived from iPS cells, we have identified CD82, a member of the tetraspanin family. CD82 + cells at the EP stage differentiated into endocrine cells more efficiently than CD82 − EP stage cells. We also show that CD82 + cells in human islets secreted insulin more efficiently than CD82 − cells. Furthermore, knockdown of CD82 expression by siRNA or inhibition of CD82 by monoclonal antibodies in NGN3 + cells suppressed the function of β cells with glucose-stimulated insulin secretion, suggesting that CD82 plays a role in maturation of EP cells to β cells.