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Two independent systems impairing hepatocyte proliferation during liver injury cause physiologically significant levels of functional hepatocyte regeneration from biliary cells. Bile duct sends on substitutes for injured liver Liver injury triggers regeneration through proliferation of hepatocytes to compensate for the loss of cells. However, in humans, this process is impaired in chronic liver diseases and it has been difficult to reproduce this impairment in mouse models of liver injury. Stuart Forbes and colleagues have used two systems to impair hepatocyte proliferation, via impairment of β-integrin or overexpression of the cell cycle inhibitor p21. They observe that, following injury under these conditions, epithelial bile duct cells are able to adopt hepatic fate and regenerate up to 25% of functional hepatocytes. After liver injury, regeneration occurs through self-replication of hepatocytes. In severe liver injury, hepatocyte proliferation is impaired—a feature of human chronic liver disease 1 , 2 . It is unclear whether other liver cell types can regenerate hepatocytes 3 , 4 , 5 . Here we use two independent systems to impair hepatocyte proliferation during liver injury to evaluate the contribution of non-hepatocytes to parenchymal regeneration. First, loss of β1-integrin in hepatocytes with liver injury triggered a ductular reaction of cholangiocyte origin, with approximately 25% of hepatocytes being derived from a non-hepatocyte origin. Second, cholangiocytes were lineage traced with concurrent inhibition of hepatocyte proliferation by β1-integrin knockdown or p21 overexpression, resulting in the significant emergence of cholangiocyte-derived hepatocytes. We describe a model of combined liver injury and inhibition of hepatocyte proliferation that causes physiologically significant levels of regeneration of functional hepatocytes from biliary cells.

The Notch signalling pathway is an evolutionarily conserved mechanism of cell–cell communication that mediates cellular proliferation, fate determination and maintenance of stem/progenitor cell populations across tissues. Although it was originally identified as a critical regulator of embryonic liver development, NOTCH signalling activation has been associated with the pathogenesis of a number of paediatric and adult liver diseases. It remains unclear, however, what role NOTCH actually plays in these pathophysiological processes and whether NOTCH activity represents the reactivation of a conserved developmental programme that is essential for adult tissue repair. In this Review, we explore the concepts that NOTCH signalling reactivation in the biliary epithelium is a reiterative and essential response to bile duct damage and that, in disease contexts in which biliary epithelial cells need to be regenerated, NOTCH signalling supports ductular regrowth. Furthermore, we evaluate the recent literature on NOTCH signalling as a critical factor in progenitor-mediated hepatocyte regeneration, which indicates that the mitogenic role for NOTCH signalling in biliary epithelial cell proliferation has also been co-opted to support other forms of epithelial regeneration in the adult liver.

Cellular senescence is a mechanism that provides an irreversible barrier to cell cycle progression to prevent undesired proliferation. However, under pathological circumstances, senescence can adversely affect organ function, viability and regeneration. We have developed a mouse model of biliary senescence, based on the conditional deletion of Mdm2 in bile ducts under the control of the Krt19 promoter, that exhibits features of biliary disease. Here we report that senescent cholangiocytes induce profound alterations in the cellular and signalling microenvironment, with recruitment of myofibroblasts and macrophages causing collagen deposition, TGFβ production and induction of senescence in surrounding cholangiocytes and hepatocytes. Finally, we study how inhibition of TGFβ-signalling disrupts the transmission of senescence and restores liver function. We identify cellular senescence as a detrimental mechanism in the development of biliary injury. Our results identify TGFβ as a potential therapeutic target to limit senescence-dependent aggravation in human cholangiopathies. Senescence has been suggested as causing biliary cholangiopathies but how this is regulated is unclear. Here, the authors generate a mouse model of biliary senescence by deleting Mdm2 in bile ducts and show that inhibiting TGFβ limits senescence-dependent aggravation of cholangiopathies.

Hepatic precursor cells (HPCs) are known to be bipotent and to give rise to both new hepatocytes and cholangiocytes upon acute liver injury. Stuart J. Forbes and his colleagues now show that interactions of HPCs with local macrophages and myofibroblasts potentiate Wnt and Notch signaling, respectively, to determine fate specification of the HPCs. Together, these mechanisms help determine proper organ regeneration after liver injury. During chronic injury a population of bipotent hepatic progenitor cells (HPCs) become activated to regenerate both cholangiocytes and hepatocytes. Here we show in human diseased liver and mouse models of the ductular reaction that Notch and Wnt signaling direct specification of HPCs via their interactions with activated myofibroblasts or macrophages. In particular, we found that during biliary regeneration, expression of Jagged 1 (a Notch ligand) by myofibroblasts promoted Notch signaling in HPCs and thus their biliary specification to cholangiocytes. Alternatively, during hepatocyte regeneration, macrophage engulfment of hepatocyte debris induced Wnt3a expression. This resulted in canonical Wnt signaling in nearby HPCs, thus maintaining expression of Numb (a cell fate determinant) within these cells and the promotion of their specification to hepatocytes. By these two pathways adult parenchymal regeneration during chronic liver injury is promoted.

Although macrophages are widely recognized to have a profibrotic role in inflammation, we have used a highly tractable CCl 4 -induced model of reversible hepatic fibrosis to identify and characterize the macrophage phenotype responsible for tissue remodeling: the hitherto elusive restorative macrophage. This CD11B hi F4/80 int Ly-6C lo macrophage subset was most abundant in livers during maximal fibrosis resolution and represented the principle matrix metalloproteinase (MMP) -expressing subset. Depletion of this population in CD11B promoter–diphtheria toxin receptor (CD11B-DTR) transgenic mice caused a failure of scar remodeling. Adoptive transfer and in situ labeling experiments showed that these restorative macrophages derive from recruited Ly-6C hi monocytes, a common origin with profibrotic Ly-6C hi macrophages, indicative of a phenotypic switch in vivo conferring proresolution properties. Microarray profiling of the Ly-6C lo subset, compared with Ly-6C hi macrophages, showed a phenotype outside the M1/M2 classification, with increased expression of MMPs, growth factors, and phagocytosis-related genes, including Mmp9, Mmp12, insulin-like growth factor 1 (Igf1), and Glycoprotein (transmembrane) nmb (Gpnmb). Confocal microscopy confirmed the postphagocytic nature of restorative macrophages. Furthermore, the restorative macrophage phenotype was recapitulated in vitro by the phagocytosis of cellular debris with associated activation of the ERK signaling cascade. Critically, induced phagocytic behavior in vivo, through administration of liposomes, increased restorative macrophage number and accelerated fibrosis resolution, offering a therapeutic strategy to this orphan pathological process.

The liver is a complex organ that requires multiple rounds of cell fate decision for development and homeostasis throughout the lifetime. During the earliest phases of organogenesis, the liver acquires a separate lineage from the pancreas and the intestine, and subsequently, the liver bud must appropriately differentiate to form metabolic hepatocytes and cholangiocytes for proper hepatic physiology. In addition, throughout life, the liver is bombarded with chemical and pathological insults, which require the activation and correct differentiation of adult progenitor cells. This Review seeks to provide an overview of the complex signaling relationships that allow these tightly regulated processes to occur.

The Notch signaling pathway is an evolutionarily conserved mechanism of cell–cell communication that mediates cellular proliferation, cell fate specification, and maintenance of stem and progenitor cell populations. In the vertebrate liver, an absence of Notch signaling results in failure to form bile ducts, a complex tubular network that radiates throughout the liver, which, in healthy individuals, transports bile from the liver into the bowel. Loss of a functional biliary network through congenital malformations during development results in cholestasis and necessitates liver transplantation. Here, we examine to what extent Notch signaling is necessary throughout embryonic life to initiate the proliferation and specification of biliary cells and concentrate on the animal and human models that have been used to define how perturbations in this signaling pathway result in developmental liver disorders.

Cholangiocarcinoma (CCA) is a diverse collection of malignant tumors that originate in the bile ducts. Mitochondria, the energy converters in eukaryotic cells, contain circular mitochondrial DNA (mtDNA) which has a greater mutation rate than nuclear DNA. Heteroplasmic variations in mtDNA may suggest an increased risk of cancer-related mortality, serving as a potential prognostic marker. In this study, we investigated the mtDNA variations of five CCA cell lines, including KKU-023, KKU-055, KKU-100, KKU213A, and KKU-452 and compared them to the non-tumor cholangiocyte MMNK-1 cell line. We used Oxford Nanopore Technologies (ONT), a long-read sequencing technology capable of synthesizing the whole mitochondrial genome, which facilitates enhanced identification of complicated rearrangements in mitogenomics. The analysis revealed a high frequency of SNVs and INDELs, particularly in the D-loop, MT-RNR2 , MT-CO1 , MT-ND4 , and MT-ND5 genes. Significant mutations were detected in all CCA cell lines, with particularly notable non-synonymous SNVs such as m.8462T > C in KKU-023, m.9493G > A in KKU-055, m.9172C > A in KKU-100, m.15024G > C in KKU-213A, m.12994G > A in KKU-452, and m.13406G > A in MMNK-1, which demonstrated high pathogenicity scores. The presence of these mutations suggests the potential for mitochondrial dysfunction and CCA progression. Analysis of mtDNA structural variants (SV) revealed significant variability among the cell lines. We identified 208 SVs in KKU-023, 185 SVs in KKU-055, 231 SVs in KKU-100, 69 SVs in KKU-213A, 172 SVs in KKU-452, and 217 SVs in MMNK-1. These SVs included deletions, duplications, and inversions, with the highest variability observed in KKU-100 and the lowest in KKU-213A. Our results underscore the diverse mtDNA mutation landscape in CCA cell lines, highlighting the potential impact of these mutations on mitochondrial function and CCA cell line progression. Future research is required to investigate the functional impacts of these variants, their interactions with nuclear DNA in CCA, and their potential as targets for therapeutic intervention.

Cholangiocarcinoma (CCA) is a diverse collection of malignant tumors that originate in the bile ducts. Mitochondria, the energy converters in eukaryotic cells, contain circular mitochondrial DNA (mtDNA) which has a greater mutation rate than nuclear DNA. Heteroplasmic variations in mtDNA may suggest an increased risk of cancer-related mortality, serving as a potential prognostic marker. In this study, we investigated the mtDNA variations of five CCA cell lines, including KKU-023, KKU-055, KKU-100, KKU213A, and KKU-452 and compared them to the non-tumor cholangiocyte MMNK-1 cell line. We used Oxford Nanopore Technologies (ONT), a long-read sequencing technology capable of synthesizing the whole mitochondrial genome, which facilitates enhanced identification of complicated rearrangements in mitogenomics. The analysis revealed a high frequency of SNVs and INDELs, particularly in the D-loop, MT-RNR2, MT-CO1, MT-ND4, and MT-ND5 genes. Significant mutations were detected in all CCA cell lines, with particularly notable non-synonymous SNVs such as m.8462T > C in KKU-023, m.9493G > A in KKU-055, m.9172C > A in KKU-100, m.15024G > C in KKU-213A, m.12994G > A in KKU-452, and m.13406G > A in MMNK-1, which demonstrated high pathogenicity scores. The presence of these mutations suggests the potential for mitochondrial dysfunction and CCA progression. Analysis of mtDNA structural variants (SV) revealed significant variability among the cell lines. We identified 208 SVs in KKU-023, 185 SVs in KKU-055, 231 SVs in KKU-100, 69 SVs in KKU-213A, 172 SVs in KKU-452, and 217 SVs in MMNK-1. These SVs included deletions, duplications, and inversions, with the highest variability observed in KKU-100 and the lowest in KKU-213A. Our results underscore the diverse mtDNA mutation landscape in CCA cell lines, highlighting the potential impact of these mutations on mitochondrial function and CCA cell line progression. Future research is required to investigate the functional impacts of these variants, their interactions with nuclear DNA in CCA, and their potential as targets for therapeutic intervention.