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Abstract Background Liver transplantation remains the only curative treatment for end-stage liver diseases. Unfortunately, there is a drastic organ donor shortage. Hepatocyte transplantation emerged as a viable alternative to liver transplantation. Considering their unique expansion capabilities and their potency to be driven toward a chosen cell fate, pluripotent stem cells are extensively studied as an unlimited cell source of hepatocytes for cell therapy. It has been previously shown that freshly prepared hepatocyte-like cells can cure mice from acute and chronic liver failure and restore liver function. Methods Human PSC-derived immature hepatic progenitors (GStemHep) were generated using a new protocol with current good manufacturing practice compliant conditions from PSC amplification and hepatic differentiation to cell cryopreservation. The therapeutic potential of these cryopreserved cells was assessed in two clinically relevant models of acute liver failure, and the mode of action was studied by several analytical methods, including unbiased proteomic analyses. Results GStemHep cells present an immature hepatic phenotype (alpha-fetoprotein positive, albumin negative), secrete hepatocyte growth factor and do not express major histocompatibility complex. A single dose of thawed GStemHep rescue mice from sudden death caused by acetaminophen and thioacetamide-induced acute liver failure, both in immunodeficient and immunocompetent animals in the absence of immunosuppression. Therapeutic biological effects were observed as soon as 3 h post-cell transplantation with a reduction in serum transaminases and in liver necrosis. The swiftness of the therapeutic effect suggests a paracrine mechanism of action of GStemHep leading to a rapid reduction of inflammation as well as a rapid cytoprotective effect with as a result a proteome reprograming of the host hepatocytes. The mode of action of GStemHep relie on the alleviation of inhibitory factors of liver regeneration, an increase in proliferation-promoting factors and a decrease in liver inflammation. Conclusions We generated cryopreserved and current good manufacturing practice-compliant human pluripotent stem cell-derived immature hepatic progenitors that were highly effective in treating acute liver failure through rapid paracrine effects reprogramming endogenous hepatocytes. This is also the first report highlighting that human allogeneic cells could be used as cryopreserved cells and in the absence of immunosuppression for human PSC-based regenerative medicine for acute liver failure.

Background: Liver transplantation remains the only curative treatment for end-stage liver diseases. Unfortunately, there is a drastic organ donor shortage. Hepatocyte transplantation emerged as a viable alternative to liver transplantation. Considering their unique expansion capabilities and their potency to be driven toward a chosen cell fate, pluripotent stem cells are extensively studied as an unlimited cell source of hepatocytes for cell therapy. It has been previously shown that freshly prepared hepatocyte-like cells can cure mice from acute and chronic liver failure and restore liver function. Methods: Human PSC-derived immature hepatic progenitors (GStemHep) were generated using a new protocol with current good manufacturing practice compliant conditions from PSC amplification and hepatic differentiation to cell cryopreservation. The therapeutic potential of these cryopreserved cells was assessed in two clinically relevant models of acute liver failure, and the mode of action was studied by several analytical methods, including unbiased proteomic analyses. Results: GStemHep cells present an immature hepatic phenotype (alpha-fetoprotein positive, albumin negative), secrete hepatocyte growth factor and do not express major histocompatibility complex. A single dose of thawed GStemHep rescue mice from sudden death caused by acetaminophen and thioacetamide-induced acute liver failure, both in immunodeficient and immunocompetent animals in the absence of immunosuppression. Therapeutic biological effects were observed as soon as 3 h post-cell transplantation with a reduction in serum transaminases and in liver necrosis. The swiftness of the therapeutic effect suggests a paracrine mechanism of action of GStemHep leading to a rapid reduction of inflammation as well as a rapid cytoprotective effect with as a result a proteome reprograming of the host hepatocytes. The mode of action of GStemHep relie on the alleviation of inhibitory factors of liver regeneration, an increase in proliferation-promoting factors and a decrease in liver inflammation. Conclusions: We generated cryopreserved and current good manufacturing practice-compliant human pluripotent stem cell-derived immature hepatic progenitors that were highly effective in treating acute liver failure through rapid paracrine effects reprogramming endogenous hepatocytes. This is also the first report highlighting that human allogeneic cells could be used as cryopreserved cells and in the absence of immunosuppression for human PSC-based regenerative medicine for acute liver failure. [ABSTRACT FROM AUTHOR]

Background: Hepatic progenitor cell (HPC) activity and regenerative process that follows pediatric acute liver failure (PALF) is still not well understood. This clinicopathological study was thus conducted with an aim to study the correlation of liver histology and HPC activity with outcomes in PALF. Methods: All PALF patients with available hepatic histological specimens were included and specimens were analyzed for hepatocyte loss, HPC activity [using cytokeratin (CK) 7, CK19, sex‐determining region Y‐related high mobility group box(SOX)9 and epithelial cell adhesion molecule (EpCAM)], hepatocyte proliferation (using Ki67), and hepatocyte senescence (using p53 and p21). Results: Ninety‐four children were included: 22 (23.4%) survived with native liver (SNL) (i.e., the good outcome group) while rest (i.e., the poor outcome group) either died [33%, 35.1%] or received liver transplant (LT) [39%, 41.5%]. When compared to subjects with poor outcomes, those in the SNL group exhibited significantly less severe hepatocyte loss, fewer HPC/hpf, more proliferating hepatocytes, and less senescent hepatocytes (p <.05). Increasing severity of hepatocyte loss (adjusted OR: 9.95, 95% CI: 4.22–23.45, p <.001) was identified as an independent predictor of poor outcome. Eighty percent children with >50% native hepatocyte loss had poor outcome within 10 days of hospitalization. Conclusion: In PALF, more severe hepatocyte loss, higher number of HPC activation, lesser number of proliferating hepatocytes, and greater number of senescent hepatocytes are associated with a poor outcome. Loss of >50% hepatocytes is an independent predictor of poor outcome in PALF. [ABSTRACT FROM AUTHOR]

The liver is the only organ that can regenerate and regain its original tissue-to-body weight ratio within a short period of time after tissue loss. Insufficient liver regeneration in patients after partial hepatectomy or liver transplantation with partial liver grafts often leads to post-hepatectomy liver failure or small-for-size syndrome, respectively. Enhancing liver regeneration after liver injury might improve outcomes and increase patient survival. Liver regeneration comprises hepatocyte proliferation, and hepatic progenitor cell expansion and differentiation into hepatocytes. The immune system is intensively involved in liver regeneration. The current review provides a comprehensive overview of the diverse roles played by immune cells in liver regeneration. Macrophages, neutrophils, eosinophils, basophils, mast cells, platelets, dendritic cells, type 1 innate lymphoid cells, B cells, and T cells are implicated in promoting liver regeneration, while natural killer cells and overactivated natural killer T cells are supposed to inhibit hepatocyte proliferation. We also highlight the predominant underlying mechanisms mediated by immune cells, which may contribute to the development of novel strategies for promoting liver regeneration in patients with liver diseases.

The liver is the only organ that can regenerate and regain its original tissue-to-body weight ratio within a short period of time after tissue loss. Insufficient liver regeneration in patients after partial hepatectomy or liver transplantation with partial liver grafts often leads to post-hepatectomy liver failure or small-for-size syndrome, respectively. Enhancing liver regeneration after liver injury might improve outcomes and increase patient survival. Liver regeneration comprises hepatocyte proliferation, and hepatic progenitor cell expansion and differentiation into hepatocytes. The immune system is intensively involved in liver regeneration. The current review provides a comprehensive overview of the diverse roles played by immune cells in liver regeneration. Macrophages, neutrophils, eosinophils, basophils, mast cells, platelets, dendritic cells, type 1 innate lymphoid cells, B cells, and T cells are implicated in promoting liver regeneration, while natural killer cells and overactivated natural killer T cells are supposed to inhibit hepatocyte proliferation. We also highlight the predominant underlying mechanisms mediated by immune cells, which may contribute to the development of novel strategies for promoting liver regeneration in patients with liver diseases. [ABSTRACT FROM AUTHOR]

The malignant transformation of hepatic progenitor cells (HPCs) in the inflammatory microenvironment is the root cause of hepatocarcinogenesis. However, the potential molecular mechanisms are still elusive. The HPCs subgroup is identified by single‐cell RNA (scRNA) sequencing and the phenotype of HPCs is investigated in the primary HCC model. Bulk RNA sequencing (RNA‐seq) and proteomic analyses are also performed on HPC‐derived organoids. It is found that tumors are formed from HPCs in peritumor tissue at the 16th week in a HCC model. Furthermore, it is confirmed that the macrophage‐derived TWEAK/Fn14 promoted the expression of inhibitor of differentiation‐1 (ID1) in HPCs via NF‐κB signaling and a high level of ID1 induced aberrant differentiation of HPCs. Mechanistically, ID1 suppressed differentiation and promoted proliferation in HPCs through the inhibition of HNF4α and Rap1GAP transcriptions. Finally, scRNA sequencing of HCC patients and investigation of clinical specimens also verified that the expression of ID1 is correlated with aberrant differentiation of HPCs into cancer stem cells, patients with high levels of ID1 in HPCs showed a poorer prognosis. This study provides important intervention targets and a theoretical basis for the clinical diagnosis and treatment of HCC. [ABSTRACT FROM AUTHOR]

Abstract Background & Aims Capsaicin, a functional component of chili pepper, possesses anti‐inflammatory, analgesic, and anti‐cancer properties. This study aimed to determine the property of capsaicin against hepatocarcinogenesis in vivo and investigate the role of the SIRT1/SOX2 pathway in the mode of action of capsaicin in hepatic progenitor cells (HPCs), which is related to hepatocarcinogenesis. Materials & Methods We prepared a diethylnitrosamine‐induced liver cancer model in rats to examine hepatocarcinogenesis, and delivered liposomal capsaicin through the subcutaneous transposition of the spleen to the liver. Liver sections from rats and hepatocarcinoma patients were stained for the markers of HPCs or SIRT1/SOX2 signaling. SIRT1/SOX2 signalling expression was measured using immunoprecipitation and western blot. Results We found that capsaicin significantly inhibited hepatocarcinogenesis. Notably, capsaicin inhibited HPCs activation in vivo but did not induce apoptosis in the normal hepatic progenitor cell line in rats in vitro. This suggests that capsaicin suppresses hepatocarcinogenesis by inhibiting the stemness of HPCs. Moreover, capsaicin can induce this inhibition by reducing the stability of SOX2. SIRT1 is overexpressed in liver cancer and acts as a tumor promoter via SOX2 deacetylation. Using immunoprecipitation, we identified direct binding between SIRT1 and SOX2. The capsaicin treatment resulted in SIRT1 downregulation which reduced deacetylation, and increased nuclear export as well as subsequent ubiquitous degradation of SOX2. Conclusions Altogether, we report that capsaicin suppresses hepatocarcinogenesis by inhibiting the stemness of HPCs via SIRT1/SOX2 signaling. It may serve as a promising therapeutic candidate for liver cancer.

Abstract The malignant transformation of hepatic progenitor cells (HPCs) in the inflammatory microenvironment is the root cause of hepatocarcinogenesis. However, the potential molecular mechanisms are still elusive. The HPCs subgroup is identified by single‐cell RNA (scRNA) sequencing and the phenotype of HPCs is investigated in the primary HCC model. Bulk RNA sequencing (RNA‐seq) and proteomic analyses are also performed on HPC‐derived organoids. It is found that tumors are formed from HPCs in peritumor tissue at the 16th week in a HCC model. Furthermore, it is confirmed that the macrophage‐derived TWEAK/Fn14 promoted the expression of inhibitor of differentiation‐1 (ID1) in HPCs via NF‐κB signaling and a high level of ID1 induced aberrant differentiation of HPCs. Mechanistically, ID1 suppressed differentiation and promoted proliferation in HPCs through the inhibition of HNF4α and Rap1GAP transcriptions. Finally, scRNA sequencing of HCC patients and investigation of clinical specimens also verified that the expression of ID1 is correlated with aberrant differentiation of HPCs into cancer stem cells, patients with high levels of ID1 in HPCs showed a poorer prognosis. This study provides important intervention targets and a theoretical basis for the clinical diagnosis and treatment of HCC.

Abstract Background The progression of Biliary Atresia (BA) is associated with the number of reactive ductular cells (RDCs) whose heterogeneity in origin and evolution in humans remains unknown. SOX9-positive liver progenitor-like cells (LPLCs) have been shown to participate in RDCs and new hepatocyte formation during cholestatic liver regeneration in an animal model, which implies the possibility that hepatocyte-reprogrammed LPLCs could be a source of RDCs in BA. The present study aimed to elucidate the characteristics of SOX9-positive LPLCs in BA for exploring new possible therapeutic targets by manipulating the bi-differentiation process of LPLCs to prevent disease progression. Methods Twenty-eight patients, including 24 patients with BA and 4 patients with Congenital Choledochal Cyst as the control group, were retrospectively recruited. Liver biopsy samples were classified histologically using a 4-point scale based on fibrosis severity. LPLCs were detected by SOX9 and HNF4A double positive staining. Single immunohistochemistry, double immunohistochemistry, and multiple immunofluorescence staining were used to determine the different cell types and characteristics of LPLCs. Results The prognostic predictors of BA, namely total bile acid (TBA), RDCs, and fibrosis, were correlated to the emergence of LPLCs. SOX9 and HNF4A double-positive LPLCs co-stained rarely with relevant markers of portal hepatic progenitor cells (portal-HPCs), including CK19, CK7, EPCAM, PROM1 (CD133), TROP2, and AFP. Under cholestasis conditions, LPLCs acquired superior proliferation and anti-senescence ability among hepatocytes. Moreover, LPLCs arranged as a pseudo-rosette structure appeared from the periportal parenchyma to the portal region, which implied the differentiation from hepatocyte-reprogrammed LPLCs to RDCs with the progression of cholestasis. Conclusions LPLCs are associated with disease progression and prognostic factors of BA. The bipotent characteristics of LPLCs are different from those of portal-HPCs. As cholestasis progresses, LPLCs appear to gain superior proliferation and anti-senescence ability and continually differentiate to RDCs.

Abstract The malignant transformation of normal resident hepatic stem/progenitor cells has a critical role in hepatocarcinogenesis and the recurrence of hepatocellular carcinoma (HCC). We defined such hepatic progenitor cells as hepatoma-initiating cells. An efficient strategy is required to target and kill the hepatoma-initiating cells. We isolated extracellular microparticles (MPs) derived from apoptotic hepatic progenitor cells (HPCs) and tested their ability to inhibit hepatocarcinogenesis. Extracellular MPs were isolated from HPCs, hepatocytes and liver tumor cells. Their effects on tumor growth were investigated in rat primary HCC models, in which hepatocarcinogenesis is induced by diethylnitrosamine (DEN). The extracellular MPs derived from apoptotic HPCs, apoptotic hepatocytes and apoptotic liver tumor cells were similar in morphology, diameter and zeta potential. However, they had different antitumor effects. In DEN-exposed rats, only the MPs derived from apoptotic HPCs effectively inhibit hepatocarcinogenesis. In vitro and in vivo analyses confirmed that HPCs preferentially take up MPs derived from apoptotic HPCs compared to MPs from other liver cell types. Proteomic analysis of MPs from apoptotic HPCs showed enrichment of proteins involved in cell death pathways. Thus, HPC-derived MPs contain a death signal to induce the killing of hepatoma-initiating cells. Our findings provide evidence that a death signal encapsulated in HPC-derived extracellular microparticles can efficiently clear hepatoma-initiating cells and prevent hepatocarcinogenesis. Graphical Abstract

Background & Aims: Capsaicin, a functional component of chili pepper, possesses anti‐inflammatory, analgesic, and anti‐cancer properties. This study aimed to determine the property of capsaicin against hepatocarcinogenesis in vivo and investigate the role of the SIRT1/SOX2 pathway in the mode of action of capsaicin in hepatic progenitor cells (HPCs), which is related to hepatocarcinogenesis. Materials & Methods: We prepared a diethylnitrosamine‐induced liver cancer model in rats to examine hepatocarcinogenesis, and delivered liposomal capsaicin through the subcutaneous transposition of the spleen to the liver. Liver sections from rats and hepatocarcinoma patients were stained for the markers of HPCs or SIRT1/SOX2 signaling. SIRT1/SOX2 signalling expression was measured using immunoprecipitation and western blot. Results: We found that capsaicin significantly inhibited hepatocarcinogenesis. Notably, capsaicin inhibited HPCs activation in vivo but did not induce apoptosis in the normal hepatic progenitor cell line in rats in vitro. This suggests that capsaicin suppresses hepatocarcinogenesis by inhibiting the stemness of HPCs. Moreover, capsaicin can induce this inhibition by reducing the stability of SOX2. SIRT1 is overexpressed in liver cancer and acts as a tumor promoter via SOX2 deacetylation. Using immunoprecipitation, we identified direct binding between SIRT1 and SOX2. The capsaicin treatment resulted in SIRT1 downregulation which reduced deacetylation, and increased nuclear export as well as subsequent ubiquitous degradation of SOX2. Conclusions: Altogether, we report that capsaicin suppresses hepatocarcinogenesis by inhibiting the stemness of HPCs via SIRT1/SOX2 signaling. It may serve as a promising therapeutic candidate for liver cancer. [ABSTRACT FROM AUTHOR]

Background: The progression of Biliary Atresia (BA) is associated with the number of reactive ductular cells (RDCs) whose heterogeneity in origin and evolution in humans remains unknown. SOX9-positive liver progenitor-like cells (LPLCs) have been shown to participate in RDCs and new hepatocyte formation during cholestatic liver regeneration in an animal model, which implies the possibility that hepatocyte-reprogrammed LPLCs could be a source of RDCs in BA. The present study aimed to elucidate the characteristics of SOX9-positive LPLCs in BA for exploring new possible therapeutic targets by manipulating the bi-differentiation process of LPLCs to prevent disease progression. Methods: Twenty-eight patients, including 24 patients with BA and 4 patients with Congenital Choledochal Cyst as the control group, were retrospectively recruited. Liver biopsy samples were classified histologically using a 4-point scale based on fibrosis severity. LPLCs were detected by SOX9 and HNF4A double positive staining. Single immunohistochemistry, double immunohistochemistry, and multiple immunofluorescence staining were used to determine the different cell types and characteristics of LPLCs. Results: The prognostic predictors of BA, namely total bile acid (TBA), RDCs, and fibrosis, were correlated to the emergence of LPLCs. SOX9 and HNF4A double-positive LPLCs co-stained rarely with relevant markers of portal hepatic progenitor cells (portal-HPCs), including CK19, CK7, EPCAM, PROM1 (CD133), TROP2, and AFP. Under cholestasis conditions, LPLCs acquired superior proliferation and anti-senescence ability among hepatocytes. Moreover, LPLCs arranged as a pseudo-rosette structure appeared from the periportal parenchyma to the portal region, which implied the differentiation from hepatocyte-reprogrammed LPLCs to RDCs with the progression of cholestasis. Conclusions: LPLCs are associated with disease progression and prognostic factors of BA. The bipotent characteristics of LPLCs are different from those of portal-HPCs. As cholestasis progresses, LPLCs appear to gain superior proliferation and anti-senescence ability and continually differentiate to RDCs. [ABSTRACT FROM AUTHOR]

Abstract Background Hepatic schistosomiasis, a chronic liver injury induced by long-term Schistosoma japonicum (S. japonicum) infection, is characterized by egg granulomas and fibrotic pathology. Hepatic progenitor cells (HPCs), which are nearly absent or quiescent in normal liver, play vital roles in chronic and severe liver injury. But their role in the progression of liver injury during infection remains unknown. Methods In this study, the hepatic egg granulomas, fibrosis and proliferation of HPCs were analyzed in the mice model of S. japonicum infection at different infectious stages. For validating the role of HPCs in hepatic injury, tumor necrosis factor-like-weak inducer of apoptosis (TWEAK) and TWEAK blocking antibody were used to manipulate the proliferation of HPCs in wild-type and IL-33−/− mice infected with S. japonicum. Results We found that the proliferation of HPCs was accompanied by inflammatory granulomas and fibrosis formation. HPCs expansion promoted liver regeneration and inhibited inflammatory egg granulomas, as well as the deposition of fibrotic collagen. Interestingly, the expression of IL-33 was negatively associated with HPCs’ expansion. There were no obvious differences of liver injury caused by infection between wild-type and IL-33−/− mice with HPCs’ expansion. However, liver injury was more attenuated in IL-33−/− mice than wild-type mice when the proliferation of HPCs was inhibited by anti-TWEAK. Conclusions Our data uncovered a protective role of HPCs in hepatic schistosomiasis in an IL-33-dependent manner, which might provide a promising progenitor cell therapy for hepatic schistosomiasis.

Objective: to obtain long-lived proliferating cells with progenitor features by dedifferentiation of mature rat hepatocytes using combinations of small molecules.Materials and Methods. Hepatocytes isolated from rat liver by perfusion were cultured in the presence of a cocktail of three small molecules – Wnt signaling pathway activator (CHIR99021), TGF-β inhibitors (A83-01) and ROCK kinase (Y27632). The morphological characteristics and growth features of the culture were assessed using fluorescence and phase-contrast microscopy during cell culture. Cell proliferative activity was analyzed using real-time time-lapse imaging. The expression of surface and intracellular markers was analyzed using flow cytometry and high-resolution fluorescence microscopy.Results. Using a cocktail of small molecules, Y-27632, A-83-01, and CHIR99021, long-lived proliferating cells that express progenitor cell markers, such as α-fetoprotein and HNF4α, were obtained from mature rat hepatocytes. The cells had hepatocyte-like morphology and formed discrete clusters of proliferating cells, forming a single cell layer during culturing. Removal of the small molecules from the medium led to expansion of fibroblast-like cells and elimination of potentially progenitor hepatocyte-like cells.Conclusion. Proliferating progenitor cells can be obtained by dedifferentiation of mature hepatocytes.

Intrahepatic cholangiocarcinoma (ICC) is a highly lethal hepatobiliary neoplasm, which originates from the bile ducts proximal to the second-order division. ICC can be anatomically divided into two subtypes: the large duct type (mucin-production ICC, muc-ICC) and the small duct type (mixed-ICC) origins from hepatic progenitor cells (HPCs). The immunoreactivity of S100P and neural cell adhesion molecule (NCAM) are useful biomarkers to distinguish the two subtypes. In this study, we report a difficult-to-diagnose case of metastatic retroperitoneal tumor of occult hepatolithiasis-associated ICC. Besides, this case was both positive for S100P and NCAM, considered as a rare muc-ICC with the HPCs features. Tumor whole exome sequencing detection results by Genetron (China) revealed that there were 41 gene mutations in this patient. The SMAD4-p.His530ThrfsTer47 and KRAS-p.Gly12Val mutation might promote the occurrence and distant metastasis of the tumor. [ABSTRACT FROM AUTHOR]

Context Our previous studies indicated that Yiguanjian decoction (YGJ) has an anti-hepatic-fibrosis effect and could regulate macrophage status. Objective To elucidate the mechanism of YGJ in regulating macrophages. Materials and methods Liver cirrhosis was induced by CCl4 for 12 weeks combined with 2-acetylaminofluorene (2-AAF) for the last 4 weeks in male Wistar rats. YGJ (3.56 mg/kg) orally administered in the last 4 weeks, and SORA (1 mg/kg) as control. In vitro, RAW264.7 cells were treated with lipopolysaccharides (LPSs) to induce macrophage polarization to the M1 phenotype, and they were co-cultured with WB-F344 cells and allocated to M group, YGJ group (2 μg/mL) and WIF-1 group (1 μg/mL) with untreated cells as control. The differentiation direction of WB-F344 cell line was observed in the presence or absence of YGJ. Pathology, fibrosis-related cytokines, macrophage polarization-related components, and Wnt signalling pathway components were detected. Results In vivo, the expression levels of α-SMA, Col (1), OV6, SOX9, EpCAM and M1 macrophage-related components (STAT1, IRF3, IRF5, IRF8, SOCS3) significantly decreased in the YGJ group compared with those in the 2-AAF/CCl4 group (p

Intrahepatic cholangiocarcinoma (ICC) is a highly lethal hepatobiliary neoplasm, which originates from the bile ducts proximal to the second-order division. ICC can be anatomically divided into two subtypes: the large duct type (mucin-production ICC, muc-ICC) and the small duct type (mixed-ICC) origins from hepatic progenitor cells (HPCs). The immunoreactivity of S100P and neural cell adhesion molecule (NCAM) are useful biomarkers to distinguish the two subtypes. In this study, we report a difficult-to-diagnose case of metastatic retroperitoneal tumor of occult hepatolithiasis-associated ICC. Besides, this case was both positive for S100P and NCAM, considered as a rare muc-ICC with the HPCs features. Tumor whole exome sequencing detection results by Genetron (China) revealed that there were 41 gene mutations in this patient. The SMAD4-p.His530ThrfsTer47 and KRAS-p.Gly12Val mutation might promote the occurrence and distant metastasis of the tumor.

The malignant transformation of normal resident hepatic stem/progenitor cells has a critical role in hepatocarcinogenesis and the recurrence of hepatocellular carcinoma (HCC). We defined such hepatic progenitor cells as hepatoma-initiating cells. An efficient strategy is required to target and kill the hepatoma-initiating cells. We isolated extracellular microparticles (MPs) derived from apoptotic hepatic progenitor cells (HPCs) and tested their ability to inhibit hepatocarcinogenesis. Extracellular MPs were isolated from HPCs, hepatocytes and liver tumor cells. Their effects on tumor growth were investigated in rat primary HCC models, in which hepatocarcinogenesis is induced by diethylnitrosamine (DEN). The extracellular MPs derived from apoptotic HPCs, apoptotic hepatocytes and apoptotic liver tumor cells were similar in morphology, diameter and zeta potential. However, they had different antitumor effects. In DEN-exposed rats, only the MPs derived from apoptotic HPCs effectively inhibit hepatocarcinogenesis. In vitro and in vivo analyses confirmed that HPCs preferentially take up MPs derived from apoptotic HPCs compared to MPs from other liver cell types. Proteomic analysis of MPs from apoptotic HPCs showed enrichment of proteins involved in cell death pathways. Thus, HPC-derived MPs contain a death signal to induce the killing of hepatoma-initiating cells. Our findings provide evidence that a death signal encapsulated in HPC-derived extracellular microparticles can efficiently clear hepatoma-initiating cells and prevent hepatocarcinogenesis. [ABSTRACT FROM AUTHOR]

Our previous studies indicated that Yiguanjian decoction (YGJ) has an anti-hepatic-fibrosis effect and could regulate macrophage status. To elucidate the mechanism of YGJ in regulating macrophages. Liver cirrhosis was induced by CCl4 for 12 weeks combined with 2-acetylaminofluorene (2-AAF) for the last 4 weeks in male Wistar rats. YGJ (3.56 mg/kg) orally administered in the last 4 weeks, and SORA (1 mg/kg) as control. In vitro, RAW264.7 cells were treated with lipopolysaccharides (LPSs) to induce macrophage polarization to the M1 phenotype, and they were co-cultured with WB-F344 cells and allocated to M group, YGJ group (2 μg/mL) and WIF-1 group (1 μg/mL) with untreated cells as control. The differentiation direction of WB-F344 cell line was observed in the presence or absence of YGJ. Pathology, fibrosis-related cytokines, macrophage polarization-related components, and Wnt signalling pathway components were detected. In vivo, the expression levels of α-SMA, Col (1), OV6, SOX9, EpCAM and M1 macrophage-related components (STAT1, IRF3, IRF5, IRF8, SOCS3) significantly decreased in the YGJ group compared with those in the 2-AAF/CCl4 group (p < 0.01 or 0.05). In vitro, the expression levels of M1 macrophage-related components, including STAT1, NF-κB, IRF3, IRF5, and SOCS3, in RAW264.7 cells decreased significantly in the YGJ group compared with those in the M group (p < 0.05 or p < 0.01). The expression levels of Wnt3A, FZD5, LRP-5/-6, and β-catenin significantly increased in the YGJ group compared with those in the M group (p < 0.05 or p < 0.01). In addition, the expression levels of Wnt-4/-5A/-5B, and FZD2 significantly decreased in the YGJ group compared with those in the M group (p < 0.05 or p < 0.01). This study suggests that the anti-cirrhosis effect of YGJ is associated with its ability to inhibit macrophage M1-polarization, which provides a scientific basis for the clinical application of YGJ. [ABSTRACT FROM AUTHOR]

Abstract Lineage tracing is the most widely used technique to track the migration, proliferation, and differentiation of specific cells in vivo. The currently available gene-targeting technologies have been developing for decades to study organogenesis, tissue injury repairing, and tumor progression by tracing the fates of individual cells. Recently, lineage tracing has expanded the platforms available for disease model establishment, drug screening, cell plasticity research, and personalized medicine development in a molecular and cellular biology perspective. Lineage tracing provides new views for exploring digestive organ development and regeneration and techniques for digestive disease causes and progression. This review focuses on the lineage tracing technology and its application in digestive diseases.

Abstract Background As a critical cellular component in the hepatic stem cell niche, hepatic stellate cells (HSCs) play critical roles in regulating the expansion of hepatic stem cells, liver regeneration, and fibrogenesis. However, the signaling of HSCs, particularly that involved in promoting hepatic stem cell expansion, remains unclear. While the overexpression of galectins has been identified in regenerating liver tissues, their involvement in cell-cell interactions between HSCs and hepatic stem cells remains to be elucidated. Methods To generate a liver regeneration rat model and establish a hepatic oval cell microenvironment as a stem cell niche, 2-acetylaminofluorene treatment plus partial hepatectomy was performed. Immunofluorescence staining was conducted to detect the emergence of hepatic stem cells and their niche. Liver parenchymal cells, non-parenchymal cells, and HSCs were isolated for gene and protein expression analysis by qPCR or western blotting. To evaluate the effect of galectins on the colony-forming efficiency of hepatic stem cells, c-Kit−CD29+CD49f+/lowCD45−Ter-119− cells were cultured with recombinant galectin protein, galectin antibody, galectin-producing HSCs, and galectin-knockdown HSCs. Results Following liver injury, the cytokeratin 19+ ductal cells were robustly induced together with the emergence of OV6+CD44+CD133+EpCAM+ hepatic stem cells. The activated desmin+ HSCs were recruited around the periportal area and markedly enriched in the galectin-positive domain compared to the other non-parenchymal cells. Notably, the HSC fraction isolated from regenerating liver was accompanied by dramatically elevated gene and protein expression of galectins. Hepatic stem cells co-cultured with HSCs significantly enhanced colony-forming efficiency. Conversely, single or double knockdown of galectin-1 and galectin-3 led into a significant function loss, impaired the co-cultured hepatic stem cells to attenuated colony size, inhibited colony frequency, and reduced total cell numbers in colonies. On the other hand, the promotive function of galectins was further confirmed by recombinant galectin protein supplementation and galectins blocking antibodies. Conclusions Our findings, for the first time, demonstrated that galectins from activated HSCs contribute to hepatic stem cell expansion during liver regeneration, suggesting that galectins serve as important stem cell niche components.

Non-alcoholic fatty liver disease (NAFLD) has developed into the world's largest chronic epidemic. In NAFLD, hepatic steatosis causes hepatocytes dysfunction and even apoptosis. The liver has a strong restoration or regeneration ability after an injury, however, it is unclear through which pattern fatty liver injury in NAFLD is repaired and what the repair mechanism is. Here, we found that in the high-fat diet (HFD)-induced NAFLD mice model, fatty liver injury caused the significant ductular reaction (DR), which is a marker to promote the repair of liver injury. SOX9+ and HNF4α+ biphenotype also suggested that hepatic progenitor cells (HPCs) were activated by fatty liver injury in the HFD-elicited NAFLD mice model. Concurrently, fatty liver injury also activated the Wnt/β-catenin signal pathway, which is a necessary process for HPC differentiation into mature hepatocytes. However, Sirt1 knockdown weakened HPC activation and Wnt/β-catenin signal in Sirt1+/− mice with HFD feeding. In rat-derived WB-F344 hepatic stem cell line, Sirt1 overexpression (OE) or Sirt1 activator–Resveratrol promoted HPC differentiation via activating Wnt/β-catenin signal pathway. Glycogen PAS staining demonstrated that Sirt1 OE promoted WB-F344 cells to differentiate into mature hepatocytes with glycogen synthesis ability, while Sirt1 inhibitor EX527 or Wnt/β-catenin pathway inhibitor HF535 decreased glycogen positive cells. Together, our data suggested that Sirt1 plays a vital role in activating HPCs to repair fatty liver injury or promote liver regeneration through the Wnt/β-catenin signal pathway in NAFLD, which might provide a new strategy for fatty liver injury or NAFLD therapy.

Hepatic progenitor cells (HPCs) have a bidirectional potential to differentiate into hepatocytes and bile duct epithelial cells and constitute a second barrier to liver regeneration in the adult liver. They are usually located in the Hering duct in the portal vein region where various cells, extracellular matrix, cytokines, and communication signals together constitute the niche of HPCs in homeostasis to maintain cellular plasticity. In various types of liver injury, different cellular signaling streams crosstalk with each other and point to the inducible transcription factor set, including FoxA1/2/3, YB-1, Foxl1, Sox9, HNF4α, HNF1α, and HNF1β. These transcription factors exert different functions by binding to specific target genes, and their products often interact with each other, with diverse cascades of regulation in different molecular events that are essential for homeostatic regulation, self-renewal, proliferation, and selective differentiation of HPCs. Furthermore, the tumor predisposition of adult HPCs is found to be significantly increased under transcriptional factor dysregulation in transcriptional analysis, and the altered initial commitment of the differentiation pathway of HPCs may be one of the sources of intrahepatic tumors. Related transcription factors such as HNF4α and HNF1 are expected to be future targets for tumor treatment., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Background: Hepatic schistosomiasis, a chronic liver injury induced by long-term Schistosoma japonicum (S. japonicum) infection, is characterized by egg granulomas and fibrotic pathology. Hepatic progenitor cells (HPCs), which are nearly absent or quiescent in normal liver, play vital roles in chronic and severe liver injury. But their role in the progression of liver injury during infection remains unknown. Methods: In this study, the hepatic egg granulomas, fibrosis and proliferation of HPCs were analyzed in the mice model of S. japonicum infection at different infectious stages. For validating the role of HPCs in hepatic injury, tumor necrosis factor-like-weak inducer of apoptosis (TWEAK) and TWEAK blocking antibody were used to manipulate the proliferation of HPCs in wild-type and IL-33−/− mice infected with S. japonicum. Results: We found that the proliferation of HPCs was accompanied by inflammatory granulomas and fibrosis formation. HPCs expansion promoted liver regeneration and inhibited inflammatory egg granulomas, as well as the deposition of fibrotic collagen. Interestingly, the expression of IL-33 was negatively associated with HPCs' expansion. There were no obvious differences of liver injury caused by infection between wild-type and IL-33−/− mice with HPCs' expansion. However, liver injury was more attenuated in IL-33−/− mice than wild-type mice when the proliferation of HPCs was inhibited by anti-TWEAK. Conclusions: Our data uncovered a protective role of HPCs in hepatic schistosomiasis in an IL-33-dependent manner, which might provide a promising progenitor cell therapy for hepatic schistosomiasis. [ABSTRACT FROM AUTHOR]

Hepatocellular carcinoma (HCC) usually occurs at the late stage of chronic liver injury. Oncostatin M (OSM) is a tumor-associated cytokine highly expressed in cirrhosis and HCC patients; however, its role in hepatocarcinogenesis has not been clearly elucidated. In this study, we investigated the effect of OSM on HCC occurrence in a rat model of N-diethylnitrosamine-induced HCC. OSM overexpression significantly increased the number of tumor nodules and shortened the overall survival of the rats. Notably, OSM promoted HPC activation in vivo but did not directly regulate the proliferation of the HPC cell line in vitro. Further, OSM induced tumor necrosis factor-α (TNF-α) secretion and CD68+ macrophage accumulation, which were positively correlated with HPC activation. Additionally, TNF-α or macrophage depletion inhibited the promoting effect of OSM on hepatocarcinogenesis and HPC activation. Furthermore, OSM expression in the peritumoral tissues of HCC was positively correlated with poor overall survival of patients. In conclusion, OSM plays an important role in hepatocarcinogenesis by regulating the liver inflammation environment. Hence, OSM could be used as a potential target for HCC prevention and therapy or as an indicator of HCC prognosis. [ABSTRACT FROM AUTHOR]

The incidence of nonalcoholic fatty liver disease (NAFLD)-associated liver cancer is rising particularly in western societies. Dietary fructose has been increasingly reported as a significant contributor for the onset of NAFLD. However, the association between a high-fructose diet and liver cancer incidence is not fully understood. In this study, we investigated the effect of dietary fructose on liver tumorigenesis. In a mouse model of diethylnitrosamine (DEN)-induced liver tumor initiation, high-fructose diet was found to significantly increased hepatic lipid deposition and expression of inflammatory cytokines in mouse livers. Besides, DEN administration induced the malignant transformation of hepatic progenitor cells (HPCs) into malignant tumor initiating cells, while DEN + Fructose mice had significantly enriched HPCs with enhanced proliferation, invasion, chemoresistance, and tumorigenic capabilities. This study for the first time demonstrates that fructose promotes liver carcinogenesis via increasing tumorigenic HPCs and inflammatory microenvironment, which warrants precautionary control of the excessive intake of dietary fructose.

Objective To investigate the effect of macrophages ( MCs) on the differentiation of mouse induced pluripotent stem cells ( iPSCs) into hepatic progenitor cells ( HPCs) . Methods A total of 24 C57 BL/6 N mice were used to obtain MCs by peritoneal irrigation, and the supernatant was collected to obtain the conditioned medium of MCs ( MC - CDM) . Activin A, bone morphogenetic protein 4, and fibroblast growth factor were used to induce the differentiation of mouse iPSCs into HPCs. The differentiation of HPCs were randomly divided into control group ( normal medium) and experimental group ( MC group; use of MC - CDM medium on day 5 of induction) . Morphology, immunofluorescence assay, and Western blot were used to compare the morphology of HPCs and the expression of related proteins between the control group and the MC group. The t - test was used for comparison of continuous data between two groups. Results HPCs derived from iPSCs were established in vitro, and HPCs had the potential to differentiate into hepatocytes. Immunofluorescence assay showed that compared with the Dl2 control group, the Dl2 MC group had a significant increase in the protein expression of the HPC - specific protein CKl 9 ( 0. 90 l ± 0. 072 vs 0. 686 ± 0. 097, t = - 3. 093, P < 0. 05) . Western blot showed that compared with the Dl 2 control group, the Dl2 MC group had a significant increase in the protein expression of the HPC - related protein CK19 ( l. 922 ± 0. 103 vs l. 448 ± 0. 012, t = - 7. 881, P < 0. 05 ), as well as a significant increase in the protein expression of the autophagy - related protein LC3 ( l. 392 ± 0. 042 vs 1. 10 l ± 0. 048, t = - 5. 978, P < 0. 05 ) . Conclusion MCs can promote the differentiation of mouse iPSCs into HPCs, possibly by increasing the autophagy level of HPCs. [ABSTRACT FROM AUTHOR]

Hepatic stem cell transplantation has been demonstrated as an effective alternative therapy for the end-stage liver failure patients. Therefore, the functional detection of hepatic stem cell is essentially required. The present study confirmed that adenovirus BMP9 (Ad-BMP9) could increase the ALB-Gluc activity of HP14-19 hepatic progenitor cells, the expression of specific hepatic markers ALB, TAT, UGT1A were up-regulated while the hepatic stem cell markers DLK, AFP were down-regulated, and the number of positive Periodic acid-Schiff (PAS) stained cells were significantly higher than those in control group. However, the indocyanine green (ICG) uptake failed to be detectable in induced hepatocytes, which was inconsistent. By using another cell line LC14d, we found out that positive ICG uptake cells were located in the area of low cell density, while positive PAS stained cells were mainly concentrated in the area where cells were overlapped, indicating that different cell confluence might affect the outcomes of ICG uptake and PAS staining. A manual wound healing of Ad-BMP9 induced HP14-19 cells was made, the crawling cells were stained positive for ICG but not for PAS. Therefore, our finding may provide evidence for better application of PAS staining and ICG uptake assay in functional detection of mature hepatocytes. [ABSTRACT FROM AUTHOR]

Objectives: The final stage of liver development is the production of hepatocytes and cholangiocytes (biliary epithelial cells) from bipotent hepatic progenitor cells. We used HepaRG cells, which are bipotent and able to differentiate into both hepatocytes and cholangiocytes, as a model to study the action of a novel lncRNA (lnc‐RHL) and its role in the regulation of bipotency leading to hepatocytes and cholangiocytes. Materials and Methods: Differentiation of HepaRG cells was assessed by marker expression and morphology which revealed their ability to differentiate into hepatocytes and cholangiocytes (modelling the behaviour of hepatoblasts in vivo). Using a qRT‐PCR and RACE, we cloned a novel lncRNA (lnc‐RHL; regulator of hepatic lineages) that is upregulated upon HepaRG differentiation. Using inducible knockdown of lnc‐RHL concurrently with differentiation, we show that lnc‐RHL is required for proper HepaRG cell differentiation resulting in diminution of the hepatocyte lineage. Results: Here, we report the discovery of lnc‐RHL, a spliced and polyadenylated 670 base lncRNA expressed from the 11q23.3 apolipoprotein gene cluster. lnc‐RHL expression is confined to hepatic lineages and is upregulated when bipotent HepaRG cells are caused to differentiate. HepaRG cells made deficient for lnc‐RHL have reduced ability to differentiate into hepatocytes, but retain their ability to differentiate into cholangiocytes. Conclusions: Deficiency for lnc‐RHL in HepaRG cells converts them from bipotent progenitor cells to unipotent progenitor cells with impaired ability to yield hepatocytes. We conclude that lnc‐RHL is a key regulator of bipotency in HepaRG cells. [ABSTRACT FROM AUTHOR]

Background: It is poorly understood what cellular types participate in ductular reaction (DR) and whether DR facilitates recovery from injury or accelerates hepatic fibrosis. The aim of this study is to gain insights into the role of hepatic progenitor cell (HPC)-originated DR during fibrotic progression. Methods: DR in liver specimens of PBC, chronic HBV infection (CHB) or NAFLD, and four rodent fibrotic models by different pathogenic processes was evaluated. Gli1 expression was inhibited in rodent models or cell culture and organoid models by AAV-sh Gli1 or treating with GANT61. Results: Severity of liver fibrosis was positively correlated with DR extent in patients with PBC, CHB or NAFLD. HPCs were activated, expanded, differentiated into reactive cholangiocytes and constituted "HPC-originated DR", accompanying with exacerbated fibrosis in rodent models of HPC activation & proliferation (CCl 4 /2-AAF-treated), Μdr2 -/- spontaneous PSC, BDL-cholestatic fibrosis or WD-fed/CCl 4 -treated NASH-fibrosis. Gli1 expression was significantly increased in enriched pathways in vivo and in vitro . Enhanced Gli1 expression was identified in KRT19 + -reactive cholangiocytes. Suppressing Gli1 expression by administration of AAV-sh Gli1 or GANT61 ameliorated HPC-originated DR and fibrotic extent. KRT19 expression was reduced after GANT61 treatment in sodium butyrate-stimulated WB-F344 cells or organoids or in cells transduced with Gli1 knockdown lentiviral vectors. In contrast, KRT19 expression was elevated after transducing Gli1 overexpression lentiviral vectors in these cells. Conclusions: During various modes of chronic injury, Gli1 acted as an important mediator of HPC activation, expansion, differentiation into reactive cholangiocytes that formed DR, and subsequently provoked hepatic fibrogenesis., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

FoxA2 is an essential transcription factor for liver organogenesis and homeostasis. Although reduced expression of FoxA2 has been associated with chronic liver diseases, hepatic progenitor cells (HPCs) that are activated in these circumstances express FoxA2. However, the functional effects and underlying mechanism of FoxA2 in HPCs are still unknown. As revealed by immunostaining, HPCs expressed FoxA2 in human cirrhotic livers and in the livers of choline‐deficient diet supplemented with ethionine (CDE) rats. Knocking down FoxA2 in HPCs isolated from CDE rats significantly increased cell proliferation and aerobic glycolysis. Moreover, gene transcription, protein expression, and the enzyme activities of hexokinase 2 (HK2) were upregulated, and blocking HK2 activities via 2‐deoxyglucose markedly reduced cell proliferation and aerobic glycolysis. Kyoto Encyclopedia of Genes and Genomes analysis revealed that FoxA2 knockdown enhanced the transcription of genes involved in the phosphatidylinositol 3‐kinase (PI3K)/protein kinase B (Akt) pathway and triggered downstream Akt phosphorylation. Blocking the PI3K/Akt pathway by Ly294002 inhibited HK2 activities, aerobic glycolysis, and cell proliferation in FoxA2‐knockdown cells. Therefore, FoxA2 plays an important role in the proliferation and inhibition of HPCs by suppressing PI3K/Akt/HK2‐regulated aerobic glycolysis. [ABSTRACT FROM AUTHOR]

Background/Aims: Viral infections, especially with rotavirus, are often considered an initiator of the pathogenesis of biliary atresia (BA). However, the mechanism by which rotavirus induces BA is still unclear. Methods: A BA mouse model was induced in newborn mice by i.p. inoculation with rhesus rotavirus within 6 h of birth. The expression of Notch pathway-associated molecules (JAG1, JAG2, Notch1, Notch2, Notch3, Notch4, DII1, DII3, and DII4) was measured by quantitative PCR and western blot analysis. Bile duct obstruction was detected by hematoxylin and eosin staining and CK-19 immunohistochemical staining. DAPT was used to inhibit the Notch pathway in vivo and in vitro. Results: In the livers of patients with BA and rotavirus-induced BA mice, the expression of JAG1 and Notch2 was significantly increased. Inhibition of the Notch pathway by DAPT in vivo ameliorated bile duct obstruction and delayed BA-induced mortality. The serum levels of inflammation cytokines (TNF-α, IL-2, IL-8, and IL-18) were reduced by inhibiting the Notch pathway. The expression of CK19, Sox9, and EpCAM was significantly increased in BA liver, while DAPT treatment decreased the expression of CK19, Sox9, and EpCAM. Conclusion: Notch activation is involved in the pathogenesis of BA by promoting the differentiation of hepatic progenitor cells into cholangiocytes.

An impaired hepatocyte proliferation during severe liver injury causes the proliferation of hepatic progenitor cells (HPCs), also called as the ductular reaction (DR). In the present study, we studied the role of key angiogenic factors in HPC-mediated DR in nonalcoholic steatohepatitis (NASH). Liver biopsies from patients with NASH (n = 14) were included in the study. Patients with NASH were divided in two groups, early and late fibrosis (based on fibrosis staging). Biopsies were used to analyze the gene expression by quantitative real-time polymerase chain reaction and immunohistochemical (IHC) staining for two markers of DR, viz, CK19 and epithelial cell adhesion molecule (EpCAM). Cocultures were performed between steatotic human umbilical vein endothelial cells (HUVECs) and LX2 and Huh7 cells. Enzyme-linked immunosorbent assays were performed to measure levels of vascular endothelial growth factor (VEGF) in coculture studies. Next, Huh7 cells were treated with VEGF, and proliferation was investigated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assays. The number of EpCAM-positive cells was analyzed by flow cytometry. Of all the angiogenic factors, the gene expression of VEGF and angiopoietin 2 (Ang2) was significantly different between patients with NASH in the early and late fibrosis groups (P < 0.05 for both). Both VEGF and Ang2 also correlated significantly with the IHC scores of CK19 and EpCAM in the study group. In the in vitro studies, VEGF levels were significantly increased when Huh7 cells were cocultured with steatotic HUVECs and LX2 cells. The proliferation and percentage of EpCAM-positive cells was increased when Huh7 cells were treated with VEGF. Our study indicates an important contribution of VEGF toward the activation of HPC-mediated regeneration and DR in NASH. [ABSTRACT FROM AUTHOR]

Hepatic ischemia-reperfusion (I/R) is a major complication of liver resection, trauma, and liver transplantation; however, liver repair after I/R in diseased liver has not been studied. The present study sought to determine the manner in which the fibrotic liver repairs itself after I/R. Liver fibrosis was established in mice by CCl4 administration for 6 wk, and then liver I/R was performed to investigate liver injury and subsequent liver repair in fibrotic and control livers. After I/R, fibrotic liver had more injury compared with nonfibrotic, control liver; however, fibrotic liver showed rapid resolution of liver necrosis and reconstruction of liver parenchyma. Marked accumulation of hepatic stellate cells and macrophages were observed specifically in the fibrotic septa in early reparative phase. Fibrotic liver had higher numbers of hepatic stellate cells, macrophages, and hepatic progenitor cells during liver recovery after I/R than did control liver, but hepatocyte proliferation was unchanged. Fibrotic liver also had significantly greater number of phagocytic macrophages than control liver. Clodronate liposome injection into fibrotic mice after I/R caused decreased macrophage accumulation and delay of liver recovery. Conversely, CSF1-Fc injection into normal mice after I/R resulted in increased macrophage accumulation and concomitant decrease in necrotic tissue during liver recovery. In conclusion, fibrotic liver clears necrotic areas and restores normal parenchyma faster than normal liver after I/R. This beneficial response appears to be directly related to the increased numbers of nonparenchymal cells, particularly phagocytic macrophages, in the fibrotic liver. NEW & NOTEWORTHY This study is the first to reveal how diseased liver recovers after ischemia-reperfusion (I/R) injury. Although it was not completely unexpected that fibrotic liver had increased hepatic injury after I/R, a novel finding was that fibrotic liver had accelerated recovery and repair compared with normal liver. Enhanced repair after I/R in fibrotic liver was associated with increased expansion of phagocytic macrophages, hepatic stellate cells, and progenitor cells. [ABSTRACT FROM AUTHOR]

Hepatocytes differentiated from induced pluripotent stem cells or stem cells have the potential to be representative in vitro models of the human liver for research as well as early safety assessment programs. However, up until now, there has been no definitive proof that differentiated hepatocytes recapitulate the phenotype and functional characteristics of primary hepatocytes from the same individual. Thus, a method for the concurrent isolation of hepatocytes and hepatic stem cells is presented here to provide the cells necessary for the evaluation of the required benchmarking. The method presented here generated high-quality hepatocytes with a purity of 94 ± 1% and a high percentage viability of 79 ± 2%. Furthermore, the hepatic stem cells isolated were found to be actively proliferating and have a purity of 98 ± 1%. Thus, these isolated cells can be used as a powerful tool for the validation of differentiated hepatocyte in vitro models. [ABSTRACT FROM AUTHOR]

Hepatic stellate cells (HSCs) play an important microenvironmental role in hepatic progenitor cells (HPCs) differentiation fate. To reveal the specific mechanism of HSCs induced by transforming growth factor β1 (TGF‐β1) signaling in HPCs differentiation process, we used Knockin and knockdown technologies induced by lentivirus to upregulate or downregulate TGF‐β1 level in mouse HSCs (mHSCs) (mHSCs‐TGF‐β1 or mHSCs‐TGF‐βR1sih3). Primary mouse HPCs (mHPCs) were isolated and were cocultured with mHSCs‐TGF‐β1 and mHSCs‐TGF‐βR1sih3 for 7 days. Differentiation of mHPCs was detected by quantitative reverse transcriptase polymerase chain reaction analysis and immunofluorence in vitro. mHPCs‐E14.5 cell lines and differently treated mHSCs were cotransplanted into mice spleens immediately after establishment of acute liver injury model for animal studies. Engraftment and differentiation of transplanted cells as well as liver function recovery were measured at the seventh day via different methods. mHSCs‐TGF‐β1 were transformed into myofibroblasts and highly expressed Jagged1, but that expression was reduced after blockage of TGF‐β1 signaling. mHPCs highly expressed downstream markers of Jagged1/Notch signaling and cholangiocyte markers (CK19, SOX9, and Hes1) after coculture with mHSCs‐TGF‐β1 in vitro. In contrast, mature hepatocyte marker (ALB) was upregulated in mHPCs in coculture conditions with mHSCs‐TGF‐βR1sih3. At the seventh day of cell transplantation assay, mHPCs‐E 14.5 engrafted and differentiated into cholangiocytes after cotransplanting with TGF‐β1‐knockin mHSCs, but the cells had a tendency to differentiate into hepatocytes when transplanted with TGF‐βR1‐knockdown mHSCs, which corresponded to in vitro studies. HSCs play an important role in regulating HPCs differentiation into cholangiocytes via the TGF‐β1/Jagged1 signaling axis. However, HPCs have a tendency to differentiate into hepatocytes after blockage of TGF‐β1 signaling in HSCs. HSCs play an important role in regulating HPCs differentiation into cholangiocytes via the TGF‐β1/Jagged1 signaling axis. However, HPCs have a tendency to differentiate into hepatocytes after blockage of TGF‐β1 signaling in HSCs. [ABSTRACT FROM AUTHOR]

Hepatic progenitor cells (HPCs) in adult liver are promising for treatment of liver diseases. A biliary‐derived HPC population in adult mice has been characterized by co‐expression of stem cell marker Sry (sex determining region Y)‐box 9 (SOX9) and biliary marker cytokeratin 7 (CK7). However, isolation of these HPCs in adult healthy liver without any selection procedures remains a big challenge in this field. Here, by establishing a simple and efficient method to isolate and expand the CK7+SOX9+HPCs in vitro as clones, we acquired a stable and largely scalable cell source. The CK7+SOX9+ progenitor cells were then further induced to differentiate into hepatocyte‐like cells with expression of mature hepatocyte markers albumin (Alb) and hepatocyte nuclear factor 4 alpha (HNF4α), both in vitro and in vivo in the presence of hepatocyte growth factor (HGF) and fibroblast growth factor 9 (FGF9). Furthermore, we found that the HPCs are highly responsive to transforming growth factor‐beta (TGF‐β) signals. Collectively, we identified and harvested a CK7+SOX9+ progenitor cell population from adult mouse liver by a simple and efficient approach. The exploration of this HPC population offers an alternative strategy of generating hepatocyte‐like cells for cell‐based therapies of acute and chronic liver disorders. [ABSTRACT FROM AUTHOR]

The widespread use of hepatocytes and cholangiocytes for regenerative medicine and tissue engineering is restricted by the limited number of hepatocytes and cholangiocytes; a simple and effective method for the expansion and differentiation of the hepatic progenitor cells (HPCs) is required. Recent studies demonstrated that mouse embryonic fibroblasts (MEFs) play an important role in supporting the proliferation of the mouse hepatic progenitor cells (mHPCs). However, the effect of direct and indirect coculture of MEFs with mHPCs on the differentiation of mHPCs is poorly studied. Herein, we show that mHPCs rapidly proliferate and form colonies in direct or indirect contact coculture with MEFs in the serum-free medium. Importantly, after direct contact coculture of the mHPCs with MEFs for 6 days, mHPCs expressed the hepatic marker albumin (ALB) and did not express the cholangiocyte marker CK19, indicating their differentiation into hepatocytes. In contrast, after indirect contact coculture of the mHPCs with MEFs for 6 days, mHPCs expressed the cholangiocyte marker CK19 and did not express the hepatic marker ALB, indicating their differentiation into cholangiocytes. These results indicate that direct and indirect contact cocultures of the mHPCs with MEFs are useful for rapidly producing hepatocytes and cholangiocytes. [ABSTRACT FROM AUTHOR]

Hepatic progenitor cells (HPCs) can be activated when the liver suffers persistent and severe damage and can differentiate into hepatocytes to maintain liver regeneration and homeostasis. However, the molecular mechanism underlying the hepatic differentiation of HPCs is unclear. Therefore, in this study, we aimed to investigate the roles of autophagy and the Wnt/β-catenin signaling pathway during hepatic differentiation of HPCs in vivo and in vitro. First, immunohistochemistry, immunofluorescence and electron microscopy showed that Atg5 and β-catenin were highly expressed in human fibrotic liver and mouse liver injury induced by feeding a 50% choline-deficient diet plus 0.15% ethionine solution in drinking water (CDE diet) for 21 days; in addition, these factors were expressed in CK19-positive HPCs. Second, Western blotting and immunofluorescence confirmed that CK19-positive HPCs incubated in differentiation medium for 7 days can differentiate into hepatocytes and that differentiated HPCs were able to take up ICG and secrete albumin and urea. Further investigation via Western blotting, immunofluorescence and electron microscopy revealed autophagy and the Wnt/β-catenin pathway to be activated during hepatic differentiation of HPCs. Next, we found that inhibiting autophagy by downregulating Atg5 gene expression impaired hepatic differentiation of HPCs and inhibited activation of the Wnt/β-catenin pathway, which was rescued by overexpression of the β-catenin gene. Moreover, downregulating β-catenin gene expression without inhibiting autophagy still impeded the differentiation of HPCs. Finally, coimmunoprecipitation demonstrated that P62 forms a complex with phosphorylated glycogen synthase kinase 3 beta (pGSK3β). Third, in mouse CDE-induced liver injury, immunohistochemistry and immunofluorescence confirmed that downregulating Atg5 gene expression inhibited autophagy, thus impeding hepatic differentiation of HPCs and inhibiting activation of the Wnt/β-catenin pathway. As observed in vitro, overexpression of β-catenin rescued this phenomenon caused by autophagy inhibition, though decreasing β-catenin levels without autophagy inhibition still impeded HPC differentiation. We also found that HPCs differentiated into hepatocytes in human fibrotic liver tissue. Collectively, these results demonstrate that autophagy promotes HPC differentiation by regulating Wnt/β-catenin signaling. Our results are the first to identify a role for autophagy in promoting the hepatic differentiation of HPCs. [ABSTRACT FROM AUTHOR]

Background/Aims Whether the hepatitis B virus (HBV) infects normal hepatic stem/progenitor cells (NSCs) and if so, whether such infections play a role in the pathogenesis of HBV-induced chronic liver disease (CLD) and/or hepatocellular carcinoma (HCC) remains to be determined. The objectives of this study were to determine whether HBV infects NSCs and whether such infections alter NSC activity in a manner likely to contribute to the development of CLD and/or HCC. Methods Liver biopsies from five hepatitis B surface antigen (HBsAg) positive patients were co-stained for HBcAg and HBx and the stem cell markers EpCAM, Oct-4 and Nanog. In addition, primary NSCs derived from healthy human livers were exposed to HBV contaminated serum in vitro. Supernatant and/or cellular HBsAg, HBcAg and HBV-DNA expression were documented over the subsequent 30 days of culture. Pro- and anti-inflammatory cytokine expression, membrane potential differences (PDs), proliferative and telomerase activities of HBV-infected NSCs were also documented. Results Markers of HBV infection were present within the NSC population of all five biopsy specimens. In vitro , HBV markers appeared within three days of exposure, peaked in expression after 10–15 days and remained positive thereafter for the duration of cell viability. There were no consistent changes in HBV-infected NSC pro- or anti-inflammatory cytokine expression, membrane PDs, proliferative or telomerase activities. Conclusions Although the results of this study need to be confirmed, they suggest that HBV infects human NSCs but in the short term, do not alter those NSC features or activities associated with CLD and/or HCC. [ABSTRACT FROM AUTHOR]

ABSTRACT Background Intrahepatic cholangiocarcinomas (ICCs) are primary liver malignancies and are the second most common type of malignancy after hepatocellular carcinoma. ICCs are heterogeneous in clinical features, genotype, and biological behavior, suggesting that ICCs can initiate in different cell lineages. Aim We investigated intrahepatic cholangiocarcinoma RBE cell lines for the markers neural cell adhesion molecule (NCAM) and c-Kit, which possess hepatic progenitor cells properties. Methods NCAM + c-Kit + cells were tested for hepatic progenitor cell properties including proliferation ability, colony formation, spheroid formation, and invasiveness in NOD/SCID mice. The Agilent Whole Human Genome Microarray Kit was used to evaluate differences in gene expression related to stem cell signaling pathways between NCAM + c-Kit + and NCAM-c-Kit- subset cells. Microarray results were further confirmed by real-time RT-PCR. Results NCAM + c-Kit + cells showed hepatic progenitor cell-like traits including the abilities to self-renew and differentiate and tumorigenicity in NOD/SCID mice. Differences were observed in the expression of 421 genes related to stem cell signaling pathways (fc ≥ 2 or fc ≤ 0.5), among which 231 genes were upregulated and 190 genes were downregulated. Conclusion NCAM + c-Kit + subset cells in RBE may have properties of hepatic progenitor cells. NCAM combined with c-Kit may be a valuable marker for isolating and purifying ICC stem/progenitor cells. [ABSTRACT FROM AUTHOR]

Rapamycin (sirolimus) is a mTOR kinase inhibitor and is widely used as an immunosuppressive drug to prevent graft rejection in organ transplantation currently. However, some recent investigations have reported that it had profibrotic effect in the progression of organ fibrosis, and its precise role in the liver fibrosis is still poorly understood. Here we showed that rapamycin upregulated connective tissue growth factor (CTGF) expression at the transcriptional level in hepatic progenitor cells (HPCs). Using lentivirus-mediated small hairpin RNA (shRNA) we demonstrated that knockdown of mTOR, Raptor, or Rictor mimicked the effect of rapamycin treatment. Mechanistically, inhibition of mTOR activity with rapamycin resulted in a hyperactive PI3K-Akt pathway, whereas this activation inhibited the expression of CTGF in HPCs. Besides, rapamycin activated the TGF-β-Smad signaling, and TGF-β receptor type I (TGFβRI) serine/threonine kinase inhibitors completely blocked the effects of rapamycin on HPCs. Moreover, Smad2 was involved in the induction of CTGF through rapamycin-activated TGF-β-Smad signaling as knockdown completely blocked CTGF induction, while knockdown of Smad4 expression partially inhibited induction, whereas Smad3 knockdown had no effect. Rapamycin also induced ROS generation and latent TGF-β activation which contributed to TGF-β-Smad signaling. In conclusion, this study demonstrates that rapamycin upregulates CTGF in HPCs and suggests that rapamycin has potential fibrotic effect in liver.

Abstract Although the regeneration of the adult liver depends on hepatic progenitor cells (HPCs), many uncertainties regarding hepatic regeneration in the injured liver remain. Trefoil factor family 1 (TFF1), a secretory protein predominantly expressed in the gastrointestinal tract, is responsible for mucosal restitution. Here, we investigated the role of TFF1 in liver regeneration using a mouse model of hepatic injury (choline-deficient ethionine-supplemented diet and carbon tetrachloride administration) and genetically engineered mice (TFF1 knockout (TFF1−/−)). Immunohistochemistry analysis of human liver samples revealed TFF1 expression in the hepatocytes close to ductular reaction and the regenerating biliary epithelium in injured liver. The number of cytokeratin 19 (CK19)-positive bile ducts was significantly decreased in the TFF1−/− mice after liver injury. Notch pathway in the TFF1−/− mice was also downregulated. HPCs in the control mice differentiated into biliary cells (CK19+/SRY HMG box 9 (SOX9)+) more frequently. In contrast, HPCs in the TFF1−/− mice more frequently differentiated into a hepatic lineage (alpha fetoprotein+/SOX9+) after acute liver damage. Hepatocyte proliferation was upregulated, and the liver weight was increased in TFF1−/− mice in response to chronic liver damage. Thus, TFF1 is responsible for liver regeneration after liver injury by promoting HPC differentiation into a biliary lineage and inhibiting HPC differentiation into a hepatic lineage. Highlights • In models of hepatic injury, the effect of TFF1-deficiency is investigated. • The TFF1-deficiency result in the impaired regeneration of the bile duct. • On the other hand, the differentiation of HPCs into hepatocytes is accelerated. • Hepatic and biliary regeneration depends on the TFF1 protein in injured liver. [ABSTRACT FROM AUTHOR]

Abstract Hepatic diseases are widespread in the world and organ transplantation is currently the only treatment for liver failure. New cell-based approaches have been considered, since stem cells may represent a possible source to treat liver diseases. Acute and chronic liver diseases are characterized by high production of reactive oxygen and nitrogen species, with consequent oxidative modifications of cellular macromolecules and alteration of signaling pathways, metabolism and cell cycle. Although considered harmful molecules, reactive species are involved in cell growth and differentiation processes, modulating the activity of transcription factors, which take part in stemness/proliferation. It is conceivable that redox balance may regulate the development of hepatic progenitor cells, function and survival in synchrony with metabolism during chronic liver diseases. This review aims to summarize diverse redox-sensitive signaling pathways involved in stem cell fate, highlighting the important role of hepatic progenitor cells as a possible source to treat end-stage liver disease for organ regeneration. [ABSTRACT FROM AUTHOR]

Hepatocellular carcinoma (HCC) is an inflammation-related disease. Tumor necrosis factor alpha (TNF-α) is an important inflammatory factor and it has been confirmed to promote tumor growth and poor prognosis of HCC. Hepatic progenitor cells (HPCs) are thought to play an important role in liver injury and repair, as well as tumorigenesis. Chronic inflammation influences HPCs activation as well as differentiation. However, the mechanism is still unclear. In our study, the rat liver cancer model was constructed by DEN treatment, TNFR2-Fc fusion protein variant (TNFR2-FcV) and TNF-α-/- rats were used to detect the role of TNF-α in liver injury and tumorigenesis. And the effect of TNF-α on HPCs activation and proliferation was investigated, and the specific molecular mechanism was explored. We found that TNF-α inhibition and deletion could reduce tumor incidence but shorten survival time by increasing apoptosis and decreasing proliferation of hepatocytes. Further analysis indicated that TNF-α knochdown cloud inhibit HPCs activation and proliferation through TNFR2/STAT3 signaling pathway. And clinically TNF-α expression was correlated to HPCs activation and HCC recurrences. Our work suggested that TNF-α played a key role in liver injury and tumorigenesis. [ABSTRACT FROM AUTHOR]

Rapamycin (sirolimus) is a mTOR kinase inhibitor and is widely used as an immunosuppressive drug to prevent graft rejection in organ transplantation currently. However, some recent investigations have reported that it had profibrotic effect in the progression of organ fibrosis, and its precise role in the liver fibrosis is still poorly understood. Here we showed that rapamycin upregulated connective tissue growth factor (CTGF) expression at the transcriptional level in hepatic progenitor cells (HPCs). Using lentivirus-mediated small hairpin RNA (shRNA) we demonstrated that knockdown of mTOR, Raptor, or Rictor mimicked the effect of rapamycin treatment. Mechanistically, inhibition of mTOR activity with rapamycin resulted in a hyperactive PI3K-Akt pathway, whereas this activation inhibited the expression of CTGF in HPCs. Besides, rapamycin activated the TGF-β-Smad signaling, and TGF-β receptor type I (TGFβRI) serine/threonine kinase inhibitors completely blocked the effects of rapamycin on HPCs. Moreover, Smad2 was involved in the induction of CTGF through rapamycin-activated TGF-β-Smad signaling as knockdown completely blocked CTGF induction, while knockdown of Smad4 expression partially inhibited induction, whereas Smad3 knockdown had no effect. Rapamycin also induced ROS generation and latent TGF-β activation which contributed to TGF-β-Smad signaling. In conclusion, this study demonstrates that rapamycin upregulates CTGF in HPCs and suggests that rapamycin has potential fibrotic effect in liver. [ABSTRACT FROM AUTHOR]

Summary: In the latter half of the 20th century, our understanding of mammalian liver regeneration was shaped by the manner of compensatory hyperplasia occurring after a partial rat liver resection. This response involves almost all hepatocytes and thus is unlikely to be the outcome of the multiple cycling of a small stem cell population. It was most intense in the outer third of lobule, the location closest to the afferent arterial blood supply. With the advent of heritable genetic labelling techniques, usually applied to mice, hitherto unrecognized hepatocytes with clonogenic potential have been discovered, contributing to homoeostatic renewal and/or regenerative responses after tissue loss. This review combines observations from cell lineage tracing studies with other data to summarize the Four proposed anatomical locations for hepatocyte stem cells: the periportal zone, the pericentral zone, a randomized distribution and finally within the intrahepatic biliary tree. As in other endodermal‐derived tissues, it appears that there are both homoeostatic stem cells and regenerative stem cells, while some normally homoeostatic stem cells can become more active to boost regeneration. [ABSTRACT FROM AUTHOR]