On the Mechanisms of Biliary Flux.

Since the late 1950s, transport of bile in the liver has been described by the "osmotic concept," according to which bile flows into the canaliculi toward the ducts, countercurrent to the blood flow in the sinusoids. However, because of the small size of canaliculi, it was so far impossible to observe, let alone to quantify this process. Still, "osmotic canalicular flow" was a sufficient and plausible explanation for the clearance characteristics of a wide variety of choleretic compounds excreted in bile. Imaging techniques have now been established that allow direct flux analysis in bile canaliculi of the intact liver in living organisms. In contrast to the prevailing osmotic concept these analyses strongly suggest that the transport of small molecules in canalicular bile is diffusion dominated, while canalicular flow is negligibly small. In contrast, with the same experimental approach, it could be shown that in the interlobular ducts, diffusion is augmented by flow. Thus, bile canaliculi can be compared to a standing water zone that is connected to a river. The seemingly subtle difference between diffusion and flow is of relevance for therapy of a wide range of liver diseases including cholestasis and NAFLD. Here, we incorporated the latest findings on canalicular solute transport, and align them with extant knowledge to present an integrated and explanatory framework of bile flux that will undoubtedly be refined further in the future.

nPTD classification: an updated classification of gastric cancer location for function preserving gastrectomy based on physiological lymphatic flow.

BACKGROUND: The correlation between tumor location and lymphatic flow distribution in gastric cancer has been previously reported, and PTD (Proximal - Transitional - Distal) classification was proposed. Our group updated and developed the nPTD classification. METHOD: We retrospectively studied gastric cancer patients who underwent the dye method sentinel node biopsy from 1993 to 2020. The inclusion criteria were a single lesion type 0 cancer of ≤5 cm in the long axis, clinically node-negative, and invasion within the proper muscle layer pathologically. In this study, the distribution of dyed lymphatic flow was evaluated for each occupied area of the tumor. RESULTS: We included 416 patients in this study. The tumors located in the watershed of the right and left gastroepiploic arteries near greater curvature had extensive lymphatic flow; therefore, a newly circular region with a diameter of 5 cm is set on the watershed of the greater curvature between P and T zone as the 'n' zone. In addition, for cancers located in the lesser P curvature, lymphatic flow to the greater curvature was not observed. Therefore, the P zone was divided into two: the lesser curvature side (PL) and the greater curvature side (PG). CONCLUSIONS: The advantage of the nPTD classification is that it provides not only proper nodal dissection but also adequate function-preserving gastrectomy. If the tumor is localized within the PL, the proximal gastrectomy resection area can be further reduced. In contrast, for cancers located in the 'n' zone, near-total gastrectomy is required because of the extensive lymphatic flow.

FGF7 is a functional niche signal required for stimulation of adult liver progenitor cells that support liver regeneration.

The liver is a unique organ with a remarkably high potential to regenerate upon injuries. In severely damaged livers where hepatocyte proliferation is impaired, facultative liver progenitor cells (LPCs) proliferate and are assumed to contribute to regeneration. An expansion of LPCs is often observed in patients with various types of liver diseases. However, the underlying mechanism of LPC activation still remains largely unknown. Here we show that a member of the fibroblast growth factor (FGF) family, FGF7, is a critical regulator of LPCs. Its expression was induced concomitantly with LPC response in the liver of mouse models as well as in the serum of patients with acute liver failure. Fgf7-deficient mice exhibited markedly depressed LPC expansion and higher mortality upon toxin-induced hepatic injury. Transgenic expression of FGF7 in vivo led to the induction of cells with characteristics of LPCs and ameliorated hepatic dysfunction. We revealed that Thy1(+) mesenchymal cells produced FGF7 and appeared in close proximity to LPCs, implicating a role for those cells as the functional LPC niche in the regenerating liver. These findings provide new insights into the cellular and molecular basis for LPC regulation and identify FGF7 as a potential therapeutic target for liver diseases.

Tissue substructure-specific deposition of the ß3-containing laminin-332 in the biliary epithelium of human and mouse livers.

Laminin is a family of basement membrane proteins, whose selective and spatiotemporal expression profiles are linked to their various functions in development, maintenance, and functional regulation of different tissues. In the liver, α1-and α5-containing laminin isoforms have been documented to be critically involved in the developmental process of the epithelial tissue of the bile duct. However, possible roles of other laminin isoforms in bile duct formation and function remain elusive. Here, we evaluated public single-cell RNA sequencing databases on human liver cells to reveal expression landscape of laminin genes, and found that genes for laminin-332 subunits were conjointly expressed in the EPCAM+ biliary epithelial cell population. Expression of the ß3 and γ2 subunit genes was restricted to biliary epithelial cells in the liver and, remarkably, showed apparent heterogeneity among them. We confirmed the heterogeneous nature of the laminin-ß3 expression in murine livers, which was firmly related to morphological substructures in the biliary epithelium. Finally, we generated the liver epithelial tissue-specific laminin- ß3 knockout mice and found that this laminin subunit was dispensable under physiological conditions. Together, our present findings have identified the ß3 subunit and the related laminin-332 isoform as useful markers and potentially important regulatory molecules for future understanding of pathophysiology in the hepatobiliary system.

The transcription factor Klf5 is essential for intrahepatic biliary epithelial tissue remodeling after cholestatic liver injury.

Under various conditions of liver injury, the intrahepatic biliary epithelium undergoes dynamic tissue expansion and remodeling, a process known as ductular reaction. Mouse models defective in inducing such a tissue-remodeling process are more susceptible to liver injury, suggesting a crucial role of this process in liver regeneration. However, the molecular mechanisms regulating the biliary epithelial cell (BEC) dynamics in the ductular reaction remain largely unclear. Here, we demonstrate that the transcription factor Krüppel-like factor 5 (Klf5) is highly enriched in mouse liver BECs and plays a key role in regulating the ductular reaction, specifically under cholestatic injury conditions. Although mice lacking Klf5 in the entire liver epithelium, including both hepatocytes and BECs (Klf5-LKO (liver epithelial-specific knockout) mice), did not exhibit any apparent phenotype in the hepatobiliary system under normal conditions, they exhibited significant defects in biliary epithelial tissue remodeling upon 3,5-diethoxycarbonyl-1,4-dihydrocollidine-induced cholangitis, concomitantly with exacerbated cholestasis and reduced survival rate. In contrast, mice lacking Klf5 solely in hepatocytes did not exhibit any such phenotypes, confirming Klf5's specific role in BECs. RNA-sequencing analyses of BECs isolated from the Klf5-LKO mouse livers revealed that the Klf5 deficiency primarily affected expression of cell cycle-related genes. Moreover, immunostaining analysis with the proliferation marker Ki67 disclosed that the Klf5-LKO mice had significantly reduced BEC proliferation levels upon injury. These results indicate that Klf5 plays a critical role in the ductular reaction and biliary epithelial tissue expansion and remodeling by inducing BEC proliferation and thereby contributing to liver regeneration.

Dysregulated YAP1/TAZ and TGF-ß signaling mediate hepatocarcinogenesis in Mob1a/1b-deficient mice.

Mps One Binder Kinase Activator (MOB)1A/1B are core components of the Hippo pathway that coactivate large tumor suppressor homolog (LATS) kinases. Mob1a/1b double deficiency in mouse liver (LMob1DKO) results in hyperplasia of oval cells and immature cholangiocytes accompanied by inflammatory cell infiltration and fibrosis. More than half of mutant mice die within 3 wk of birth. All survivors eventually develop liver cancers, particularly combined hepatocellular and cholangiocarcinomas (cHC-CCs) and intrahepatic cholangiocellular carcinomas (ICCs), and die by age 60 wk. Because this phenotype is the most severe among mutant mice lacking a Hippo signaling component, MOB1A/1B constitute the critical hub of Hippo signaling in mammalian liver. LMob1DKO liver cells show hyperproliferation, increased cell saturation density, hepatocyte dedifferentiation, enhanced epithelial-mesenchymal transition and cell migration, and elevated transforming growth factor beta(TGF-ß)2/3 production. These changes are strongly dependent on Yes-Associated Protein-1 (Yap1) and partially dependent on PDZ-binding motif (Taz) and Tgfbr2, but independent of connective tissue growth factor (Ctgf). In human liver cancers, YAP1 activation is frequent in cHC-CCs and ICCs and correlates with SMAD family member 2 activation. Drug screening revealed that antiparasitic macrocyclic lactones inhibit YAP1 activation in vitro and in vivo. Targeting YAP1/TAZ with these drugs in combination with inhibition of the TGF-ß pathway may be effective treatment for cHC-CCs and ICCs.

Stem/progenitor cells in liver regeneration.

In severely or chronically injured livers where the proliferative capacity of hepatocytes is compromised, putative stem/progenitor cells are supposed to be activated. These cells are generally characterized as biliary epithelial cell marker-positive cells that emerge ectopically in the parenchymal region of the liver, as determined by histopathological examination of various liver diseases in humans and animal models. Whereas the biliary system indeed harbors cells with stem/progenitor activity that can be defined ex vivo, genetic lineage tracing studies in mice have casted doubt on their exact contribution as the genuine stem/progenitor cell population that differentiates in situ into hepatocytes. Here, I briefly review recent advances in the characterization and certification of the stem/progenitor cells in the adult liver and discuss the ongoing and future challenges to further our understanding of the cellular basis of liver regeneration. (Hepatology 2016;64:663-668).

Intrahepatic bile ducts are developed through formation of homogeneous continuous luminal network and its dynamic rearrangement in mice.

UNLABELLED: The intrahepatic bile duct (IHBD) is a highly organized tubular structure consisting of cholangiocytes, biliary epithelial cells, which drains bile produced by hepatocytes into the duodenum. Although several models have been proposed, it remains unclear how the three-dimensional (3D) IHBD network develops during liver organogenesis. Using 3D imaging techniques, we demonstrate that the continuous luminal network of IHBDs is established by 1 week after birth. Beyond this stage, the IHBD network consists of large ducts running along portal veins (PVs) and small ductules forming a mesh-like network around PVs. By analyzing embryonic and neonatal livers, we found that newly differentiated cholangiocytes progressively form a continuous and homogeneous luminal network. Elongation of this continuous network toward the liver periphery was attenuated by a potent Notch-signaling inhibitor N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester. Subsequent to this first step, the fine homogenous network is reorganized into the mature hierarchical network consisting of large ducts and small ductules. Between E17 and E18, when the homogenous network is radically reorganized into the mature hierarchical network, bile canaliculi rapidly extend and bile flow into IHBDs may increase. When formation of bile canaliculi was blocked between E16 and E18 by a multidrug resistance protein 2 inhibitor (benzbromarone), the structural rearrangement of IHBDs was significantly suppressed. CONCLUSION: Establishment of the mature IHBD network consists of two sequential events: (1) formation of the continuous luminal network regulated by the Notch-signaling pathway and (2) dynamic rearrangement of the homogeneous network into the hierarchical network induced by increased bile flow resulting from the establishment of hepatobiliary connections. (Hepatology 2016;64:175-188).

Adaptive remodeling of the biliary architecture underlies liver homeostasis.

UNLABELLED: Serving as the center for metabolism and detoxification, the liver is inherently susceptible to a wide variety of damage imposed by toxins or chemicals. Induction of cell populations with biliary epithelial phenotypes, which include progenitor-like cells and are referred to as liver progenitor cells, is often observed in histopathological examination of various liver diseases in both human patients and animal models and has been implicated in regeneration. However, the tissue dynamics underlying this phenomenon remains largely unclear. We have developed a simple imaging technique to reveal the global and fine-scale architecture of the biliary tract spreading in the mouse liver. Using this novel method, we show that the emergence and expansion of liver progenitor cells actually reflect structural transformation of the intrahepatic biliary tree in mouse liver injury models. The biliary branches expanded their area gradually and contiguously along with the course of chronic injury. Relevant regulatory signals known to be involved in liver progenitor cell regulation, including fibroblast growth factor 7 and tumor necrosis factor-like weak inducer of apoptosis, can modulate the dynamics of the biliary epithelium in different ways. Importantly, the structural transformations of the biliary tree were diverse and corresponded well with the parenchymal injury patterns. That is, when chronic hepatocyte damage was induced in the pericentral area, the biliary branches exhibited an extended structure from the periportal area with apparent tropism toward the distant injured area. CONCLUSION: The hepatobiliary system possesses a unique and unprecedented structural flexibility and can remodel dynamically and adaptively in response to various injury conditions; this type of tissue plasticity should constitute an essential component to maintain liver homeostasis.

Liver regeneration by stem/progenitor cells.

UNLABELLED: The liver is renowned for its strong, robust regenerative capacity, employing different modes of regeneration according to type and extent of injury. The process of compensatory hypertrophy of the liver upon partial hepatectomy has been standing as a classical model for studying organ regeneration in mammals and a subject of exhaustive analyses. Meanwhile, in view of the physiological relevance for many of the human liver pathologies induced upon toxic insults or hepatitis, other injury models have recently drawn increasing attention. In those damaged livers where hepatocyte proliferation is compromised, adult liver stem/progenitor cells (LPCs) are activated and differentiate to hepatocytes and cholangiocytes, leading to functional recovery of the organ. Here, we summarize and discuss recent findings on the mechanisms underlying the regeneration process of the liver. Whereas the primary focus of this article is on those related to LPC-mediated regeneration, we also introduce topics on compensatory hypertrophy, where application of new technologies and molecular genetics approaches in mice has gained a paradigm shift. Identification of various markers for LPC populations has expedited their characterization and enabled us to examine their differentiation potential in vivo using genetic lineage-tracing approaches. Comprehensive studies regarding intercellular signaling pathways and their modes of action have succeeded in elucidating novel frameworks for the LPC-niche interaction functioning in the regenerating liver. CONCLUSION: Advancing our understanding of the cellular and molecular mechanisms for liver regeneration should provide a basis for developing therapeutic strategies to treat patients with liver disease.

Development of a high throughput system to screen compounds that revert the activated hepatic stellate cells to a quiescent-like state.

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.

The combination of the low immunohistochemical expression of peroxiredoxin 4 and perilipin 2 predicts longer survival in pancreatic ductal adenocarcinoma with peroxiredoxin 4 possibly playing a main role.

Pancreatic ductal adenocarcinoma (PDAC) is a fatal disease with poor prognosis. Therefore, indicators that can be used for the early prediction of the prognosis of PDAC are needed. Peroxiredoxin (PRDX) 4 is a secretion-type antioxidant enzyme located in the cytoplasmic endoplasmic reticulum. Recent studies have reported that it is closely related to the development and prognosis of many types of cancer. Perilipin (PLIN) 2 is a lipid droplet coating protein. The high expression of PLIN2 is known to be an indicator of some types of cancer and oxidative stress management. It is highly suggestive of the interplay between PRDX4 and PLIN2 to some degree. In this study, we collected 101 patients' clinical data and paraffin-embedded specimens with PDAC and analyzed them with immunohistochemical staining of PRDX4 and PLIN2. We found that the low expression of PRDX4 predicts longer survival and a better clinical condition in PDAC patients. Moreover, when the low expression of PRDX4 is combined with the low expression of PLIN2, the 3-year survival is significantly improved. Univariate and multivariate Cox proportional hazard analyses showed that the PRDX4 expression in PDAC was an independent prognostic factor for survival. Taken together, between PRDX4 and PLIN2, PRDX4 plays a main role in prognosis and has the potential to become a clinical prognostic indicator of PDAC.

Analysis of distribution, collection, and confirmation of capacity dependency of small extracellular vesicles toward a therapy for liver cirrhosis.

BACKGROUND: The progression of liver fibrosis leads to portal hypertension and liver dysfunction. However, no antifibrotic agents have been approved for cirrhosis to date, making them an unmet medical need. Small extracellular vesicles (sEVs) of mesenchymal stem cells (MSCs) are among these candidate agents. In this study, we investigated the effects of sEVs of MSCs, analyzed their distribution in the liver post-administration, whether their effect was dose-dependent, and whether it was possible to collect a large number of sEVs. METHODS: sEVs expressing tdTomato were generated, and their uptake into constituent liver cells was observed in vitro, as well as their sites of uptake and cells in the liver using a mouse model of liver cirrhosis. The efficiency of sEV collection using tangential flow filtration (TFF) and changes in the therapeutic effects of sEVs in a volume-dependent manner were examined. RESULTS: The sEVs of MSCs accumulated mostly in macrophages in damaged areas of the liver. In addition, the therapeutic effect of sEVs was not necessarily dose-dependent, and it reached a plateau when the dosage exceeded a certain level. Furthermore, although ultracentrifugation was commonly used to collect sEVs for research purposes, we verified that TFF could be used for efficient sEV collection and that their effectiveness is not reduced. CONCLUSION: In this study, we identified some unknown aspects regarding the dynamics, collection, and capacity dependence of sEVs. Our results provide important fundamentals for the development of therapies using sEVs and hold potential implications for the therapeutic applications of sEV-based therapies for liver cirrhosis.

Two distinct Notch signals, Delta-like 4/Notch1 and Jagged-1/Notch2, antagonistically regulate chemical hepatocarcinogenesis in mice.

Notch signaling is one of the most common drivers of carcinogenesis in many types of cancers, including hepatocellular carcinoma (HCC); however, it occasionally suppresses tumor progression. Moreover, it is virtually unknown how different sets of Notch ligands and receptors regulate the HCC development. In this study, we demonstrate that the expression of the Notch ligands, Delta-like 4 (Dll4) and Jagged-1 (Jag1), is upregulated during diethylnitrosamine-induced hepatocarcinogenesis. Dll4 is detected in the preneoplastic hepatocytes and HCC cells, but not in the normal hepatocytes, while Jag1 is expressed in the desmin-positive mesenchymal cells. Hepatocyte-specific Dll4 knockout abolishes the Notch1 signaling and suppresses the tumor progression. In contrast, Jag1 deletion induces the ectopic expression of Dll4 in hepatocytes along with the loss of Notch2 signaling, leading to the tumor progression. These results indicate that the two distinct Notch signals, Dll4/Notch1 and Jag1/Notch2, are antagonistic to each other, exerting opposite effects on HCC progression.

MKP3 mediates the cellular response to FGF8 signalling in the vertebrate limb.

The mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) and phosphatidylinositol-3-OH kinase (PI3K)/Akt pathways are involved in the regulatory mechanisms of several cellular processes including proliferation, differentiation and apoptosis. Here we show that during chick, mouse and zebrafish limb/fin development, a known MAPK/ERK regulator, Mkp3, is induced in the mesenchyme by fibroblast growth factor 8 (FGF8) signalling, through the PI3K/Akt pathway. This correlates with a high level of phosphorylated ERK in the apical ectodermal ridge (AER), where Mkp3 expression is excluded. Conversely, phosphorylated Akt is detected only in the mesenchyme. Constitutively active Mek1, as well as the downregulation of Mkp3 by small interfering RNA (siRNA), induced apoptosis in the mesenchyme. This suggests that MKP3 has a key role in mediating the proliferative, anti-apoptotic signalling of AER-derived FGF8.

The differential expression of perilipin-2 in hepatoblastoma and its association with prognosis.

Perilipin-2, a lipid droplet (LD) coating protein, has been found to be involved in cancer progression. However, its role in hepatoblastoma (HB) is undefined. We collected 87 HB samples and the corresponding clinical data. Immunohistochemistry (IHC) staining was performed to detect perilipin-2 and the association of the perilipin-2 expression with clinical characteristics and prognosis was analyzed. The expression of perilipin-2 was increased in fetal HB components in comparison to embryonal HB components. The predominant staining pattern was vesicular in fetal HB cells, while it was granular in embryonal HB cells. Furthermore, strong expression of perilipin-2 was associated with the histopathological type of fetal predominant HB. Although event-free survival (EFS) did not differ to a statistically significant extent between the strong and weak expression groups in a univariate survival analysis, a multivariate survival analysis revealed that EFS was significantly improved in the strong perilipin-2 expression group. In conclusion, perilipin-2 is differentially expressed in HB and the strong expression of perilipin-2 predicts a better prognosis.

Generation of functional liver organoids on combining hepatocytes and cholangiocytes with hepatobiliary connections ex vivo.

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.

Examination of Endoscopic Ultrasonographic Diagnosis for the Depth of Early Gastric Cancer.

BACKGROUND: Endoscopic ultrasonography (EUS) is one of the helpful tools to diagnose depth of early gastric cancer (EGC). In this study, we examined efficiencies of EUS for EGC such as overall accuracy, risk factors of over/under-staging, and accuracies of each invasive distance. METHODS: A total of 403 EGC lesions that could be investigated by EUS during pre-operation and histological diagnosis after endoscopic submucosal dissection (ESD) or surgery were enrolled in this study. For the 403 cases, we analyzed the accuracies of depth by conventional endoscopy (CE) and EUS retrospectively. We evaluated the clinical survey items of CE and EUS which will be described later to compare the differences between "accuracy group" and "over-staging group", and between "accuracy group" and "under-staging group", retrospectively. Additionally, 78 EGC lesions which were confined to the submucosa and for which it was possible to measure accurate invasive distance from the muscularis mucosae were examined for the relationship between preoperative diagnosis of depth by CE and EUS and invasive distance retrospectively. RESULTS: The overall accuracies of both CE and EUS in predicting EGC invasion depth were 87.3%. For CE staging, histological classification was the factor which influenced over-staging. Gastric regions and tumor area were the factors which influenced under-staging of CE. For EUS staging, tumor area was the factor which influenced over-staging, and gastric regions were the factors which influenced under-staging. Both CE and EUS were not sufficient for predicting the lesions confined to < 500 µm from the muscularis mucosae because the accuracies of both in predicting depth were less than 50%. However, EUS has a higher accuracy than CE for the lesions confined to 500 - 2,000 µm. CONCLUSIONS: The overall accuracies of both CE and EUS in predicting EGC invasion depth were equal, but the contributing factors for over/under-staging were different. Both CE and EUS are not sufficient at present to predict the lesions confined to < 500 µm from the muscularis mucosae. However, the accuracy of EUS in predicting them may increase if high-performance EUS systems are developed in the future.

A Case of Refractory Esophageal Ulcer Caused by Radiotherapy for Hepatocellular Carcinoma.

A 77-year-old man who underwent radiotherapy for hepatocellular carcinoma 6 months prior consulted for esophageal obstruction. Esophagogastroduodenoscopy revealed an esophageal ulcer caused by radiotherapy for hepatocellular carcinoma. He was treated with dietary counseling and vonoprazan. After 9 months, the ulcer improved but a moderate stenosis remained. Several factors such as high fraction size, history of chemotherapy, and stress associated with food intake might involve in the development of a radiation-associated ulcer. Opportunities to choose radiotherapy for hepatocellular carcinoma may increase, so we hypothesize that esophageal ulcers might be a complication that should be noted associated with this therapy.

Small extracellular vesicles derived from interferon-γ pre-conditioned mesenchymal stromal cells effectively treat liver fibrosis.

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.