Differentiation Of Hepatic Progenitor Cells Research Articles

Transcriptional Control: A Directional Sign at the Crossroads of Adult Hepatic Progenitor Cells' Fates.

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.

Review of IDH mutations and potential therapies for intrahepatic cholangiocarcinoma

Cholangiocarcinoma (CCA) is an aggressive malignancy that arises from the biliary tract. Currently, the first-line therapy for advanced CCA is gemcitabine and cisplatin. However, 5-year survival remains low. It has become abundantly clear that a “one size fits all” approach no longer applies to the treatment of individual cancers, given the large amount of tumor heterogeneity. As such, research in recent years has focused on developing effective targeted therapies through genetic profiling of CCA tumors. IDH1 and IDH2 mutations are commonly found in intrahepatic CCA (ICCA). IDH mutations prevent hepatic progenitor cell differentiation and result in the persistence of progenitor-like and stem cells. These are more prone to alterations that promote tumor initiation. As such, IDH has been identified as a promising target for ICCA treatment. We herein review the role of IDH mutations in ICCA development, recent data for IDH inhibitors in ICCA treatment, and challenges within the field of targeted therapy for ICCA.

TWEAK Signaling-Induced ID1 Expression Drives Malignant Transformation of Hepatic Progenitor Cells During Hepatocarcinogenesis.

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.

The endocannabinoid system promotes hepatocyte progenitor cell proliferation and maturation by modulating cellular energetics

The proliferation and differentiation of hepatic progenitor cells (HPCs) drive the homeostatic renewal of the liver under diverse conditions. Liver regeneration is associated with an increase in Axin2+Cnr1+ HPCs, along with a marked increase in the levels of the endocannabinoid anandamide (AEA). But the molecular mechanism linking AEA signaling to HPC proliferation and/or differentiation has not been explored. Here, we show that in vitro exposure of HPCs to AEA triggers both cell cycling and differentiation along with increased expression of Cnr1, Krt19, and Axin2. Mechanistically, we found that AEA promotes the nuclear localization of the transcription factor β-catenin, with subsequent induction of its downstream targets. Systemic analyses of cells after CRISPR-mediated knockout of the β-catenin-regulated transcriptome revealed that AEA modulates β-catenin-dependent cell cycling and differentiation, as well as interleukin pathways. Further, we found that AEA promotes OXPHOS in HPCs when amino acids and glucose are readily available as substrates, but AEA inhibits it when the cells rely primarily on fatty acid oxidation. Thus, the endocannabinoid system promotes hepatocyte renewal and maturation by stimulating the proliferation of Axin2+Cnr1+ HPCs via the β-catenin pathways while modulating the metabolic activity of their precursor cells.

Mechanism of paracrine communications between hepatic progenitor cells and endothelial cells

Hepatic progenitor cells (HPCs) are facultative tissue-specific stem cells lining reactive ductules, which are ubiquitously observed in chronic liver diseases and cancer. Although previous research mainly focused on their contribution to liver regeneration, it turned out that in vivo differentiation of HPCs into hepatocytes only occurs after extreme injury. While recent correlative evidence implies the association of HPCs with disease progression, their exact role in pathogenesis remains largely unknown. Our previous research demonstrated that HPCs expressing angiogenic paracrine factors accumulate in the peritumoral area and are positively correlated with the extent of intratumoral cell proliferation and angiogenesis in the livers of patients with liver cancer. Given the crucial roles of angiogenesis in liver disease progression and carcinogenesis, we aimed to test the hypothesis that HPCs secrete paracrine factors to communicate with endothelial cells, to determine molecular mechanisms mediating HPCs-endothelial interactions, and to understand how the paracrine function of HPCs is regulated. HPCs promoted viability and tubulogenesis of human umbilical vein endothelial cells (HUVECs) and upregulated genes known to be involved in angiogenesis, endothelial cell function, and disease progression in a paracrine manner. The paracrine function of HPCs as well as expression of colony stimulating factor 1 (CSF1) were inhibited upon differentiation of HPCs toward hepatocytes. Inhibition of CSF1 receptor partly suppressed the paracrine effects of HPCs on HUVECs. Taken together, our study indicates that inhibition of the paracrine function of HPCs through modulation of their differentiation status and inhibition of CSF1 signaling is a promising strategy for inhibition of angiogenesis during pathological progression.

Sirt1 Promotes the Restoration of Hepatic Progenitor Cell (HPC)-Mediated Liver Fatty Injury in NAFLD Through Activating the Wnt/β-Catenin Signal Pathway.

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.

Autophagy Is Required for Hepatic Differentiation of Hepatic Progenitor Cells via Wnt Signaling Pathway.

The molecular mechanisms regulating differentiation of hepatic progenitor cells (HPCs), which play pivotal roles in liver regeneration and development, remain obscure. Autophagy and Wnt signaling pathways regulate the development and differentiation of stem cells in various organs. However, the roles of autophagy and Wnt signaling pathways in hepatic differentiation of HPCs are not well understood. Here, we describe the effects of autophagy and Wnt signaling pathways during hepatic differentiation of HPCs. We used a well-established rat hepatic progenitor cell line called WB-F344, which was treated with differentiation medium to promote differentiation of WB-F344 cells along the hepatic phenotype. Firstly, autophagy was highly activated in HPCs and gradually decreased during hepatic differentiation of HPCs. Induction of autophagy by rapamycin or starvation suppressed hepatic differentiation of HPCs. Secondly, Wnt3a signaling pathway was downregulated, and Wnt5a signaling pathway was upregulated in hepatic differentiation of HPCs. At last, Wnt3a signaling pathway was enhanced, and Wnt5a signaling pathway was inhibited by activation of autophagy during hepatic differentiation of HPCs. In summary, these results demonstrate that autophagy regulates hepatic differentiation of hepatic progenitor cells through Wnt signaling pathway.

The Clinicopathological Significance of YAP/TAZ Expression in Hepatocellular Carcinoma with Relation to Hypoxia and Stemness.

Background/Aims: Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ) activation has been implicated in hepatocarcinogenesis and hepatic progenitor cell differentiation, and hypoxia has been shown to induce nuclear translocation of YAP in cancer cells. Here, we aimed to investigate the relationship between hypoxia, YAP and TAZ expression and stemness-related marker expression in human hepatocellular carcinomas (HCCs) and its clinical implications. Methods: Immunohistochemical stains were performed on tissue microarrays from 305 surgically resected HCCs, and the expression status of YAP and TAZ were correlated with CAIX, stemness markers (K19, EpCAM) and epithelial-mesenchymal transition (EMT)-related markers (uPAR, ezrin). The clinicopathological significance of YAP/TAZ expression was analyzed with relation to CAIX expression status. Results: YAP and TAZ expression were seen in 13.4 and 4.3% of HCCs, respectively. YAP/TAZ-positive HCCs frequently demonstrated higher serum AFP levels, microvascular invasion, advanced tumor stage, increased proliferative activity and expression of stemness- and EMT-related markers, CAIX, p53 and Smad2/3 (p < 0.05, all). Interestingly, YAP/TAZ-positivity was associated with microvascular invasion, higher serum AFP levels, stemness and EMT-related marker expression only in tumors expressing CAIX (p < 0.05, all), while these associations were not seen in CAIX-negative HCCs. Conclusions: YAP/TAZ expression is associated with vascular invasion, stemness and EMT in HCCs with hypoxia marker expression. The effect of Hippo signaling pathway deregulation in HCC may depend on the presence or absence of a hypoxic microenvironment, and hypoxia marker expression status should be taken into account when considering the use of YAP/TAZ as markers of aggressive biologic behavior in HCC.

Direct Effects of Transforming Growth Factor-β1 Signaling on the Differentiation Fate of Fetal Hepatic Progenitor Cells

Aim: To investigate direct roles of TGF-β1 signaling in the differentiation process of fetal hepatic progenitor cells (HPCs). Materials & methods: Exogenous TGF-β1 and SB431542 were added into fetal HPCs. Then, SB431542 was intraperitoneally injected into pregnant mice for 8 days. Results: Fetal HPCs treated with TGF-β1 differentiated into cholangiocytes. However, hepatocyte marker was highly expressed after inhibiting TGF-β1 signaling. In vivo, hematopoietic cells were gradually replaced with liver cells and TGF-β1 expression was evidently decreased as fetal liver developed. Inhibition of TGF-β1 signaling caused increase of ALB+ cells, but CK19 expression was more obvious in control mice livers. Conclusion: TGF-β1 signaling may play decisive roles in fetal HPCs differentiation into functional hepatocytes or cholangiocytes.

Effect of the transforming growth factor-β1 signaling pathway in inducing the differentiation of hepatic progenitor cells

Objective To investigate the effect of the transforming growth factor-β1( TGF-β1) signaling pathway on the differentiation of hepatic progenitor cells( HPCs). Methods HPC-E14. 5 cells were cultured in vitro and immunofluorescent staining was performed for identification. The cells were divided into control group( HPCs),HPCs + TGF-β1 group,and HPCs + SB431542 group. The cells in the HPCs + TGF-β1 group were treated with exogenous TGF-β1,and those in the HPCs + SB431542 group were treated with the TGF-β1 receptor inhibitor SB431542; after 72 hours of treatment,qRT-PCR and Western blot were used to measure the mRNA and protein expression of the stem cell marker alpha-fetoprotein( AFP),the hepatocyte marker albumin( Alb),and the cholangiocyte marker cytokeratin 19( CK19),and periodic acid-Schiff staining was used to evaluate function after differentiation. A one-way analysis of variance was used for comparison of continuous data between multiple groups,and the Dunnett's t-test was used for further comparison between two groups. Results The results of qRT-PCR showed that the control group had significantly higher mRNA expression of AFP than the other two groups( F = 128. 937,P < 0. 01),the HPCs + TGF-β1 group had significantly higher mRNA expression of CK19 than the other two groups( F =414. 467,P < 0. 01),and the HPCs + SB431542 group had the highest mRNA expression of Alb( F = 794. 929,P < 0. 01). The results of Western blot showed that the control group had significantly higher protein expression of AFP than the other two groups( F = 269. 000,P <0. 01),the HPCs + TGF-β1 group had significantly higher protein expression of CK19 than the other two groups( F = 93. 010,P < 0. 01),and the HPCs + SB431542 group had significantly higher protein expression of Alb than the other two groups( F = 1086. 000,P < 0. 01).Periodic acid-Schiff staining showed that the control group had a significantly smaller positive staining area than the other two groups( F =79. 251,P < 0. 05). Conclusion The TGF-β1 signaling pathway induces HPC differentiation into functional cholangiocytes; however,HPCs tend to differentiate into mature hepatocytes after the TGF-β1 signaling pathway is blocked. Such cholangiocytes and hepatocytes have the normal function of glycogen storage and synthesis and may participate in metabolic activities.

Conversion of mesenchymal stem cells into a canine hepatocyte-like cells by Foxa1 and Hnf4a.

IntroductionHepatocytes, which account for the majority of liver tissue, are derived from the endoderm and become hepatocytes via differentiation of hepatic progenitor cells. Induced hepatocyte-like (iHep) cells and artificial liver tissues are expected to become useful, efficient therapies for severe and refractory liver diseases and to contribute to drug discovery research. The establishment of iHep cell lines are needed to carry out liver transplants and assess liver toxicity in the rising number of dogs affected by liver disease. Recently, direct conversion of non-hepatocyte cells into iHep cells was achieved by transfecting mouse adult fibroblasts with the Forkhead box protein A1 (Foxa1) and hepatocyte nuclear factor 4 homeobox alpha (Hnf4α) genes. Here, we applied this conversion process for the differentiation of canine bone marrow stem cells (cBMSCs) into hepatocyte-like cells.MethodsBone marrow specimens were collected from four healthy Beagle dogs and used to culture cBMSCs in Dulbecco's Modified Eagle's Medium (DMEM). The cBMSCs displayed the following characteristic features: plastic adherence; differentiation into adipocytes, osteoblasts and chondrocytes; and a cell surface antigen profile of CD29 (+), CD44 (+), CD90 (+), CD45 (−), CD34 (−) and CD14 (−), or CD11b (−) and CD79a (−), or CD19 (−) and HLA class II(−). The cBMSCs were seeded in a collagen I-coated plate and cultured in DMEM with 10% fetal bovine serum and transfected with retroviruses expressing Foxa1 and Hnf4α the following day. Canine iHep cells were differentiated from cBMSCs in culture on day 10, and were analyzed for morphology, RNA expression, immunocytochemistry, urea production, and low-density lipoprotein (LDL) metabolism.ResultsThe cBMSCs expressed CD29 (98.06 ± 1.14%), CD44 (99.59 ± 0.27%) and CD90 (92.78 ± 4.89%), but did not express CD14 (0.47 ± 0.29%), CD19 (0.44 ± 0.39%), CD34 (0.33 ± 0.25%), CD45 (0.46 ± 0.34%) or MHC class II (0.54 ± 0.40%). The iHep cells exhibited morphology that included circular to equilateral circular shapes, and the formation of colonies that adhered to each other 10 days after Foxa1 and Hnf4α transfection. Quantitative RT-PCR analysis showed that the expression levels of the genes encoding albumin (ALB) and cadherin (CDH) in iHep cells on day 10 were increased approximately 100- and 10,000-fold, respectively, compared with cBMSCs. Corresponding protein expression of ALB and epithelial-CDH was confirmed by immunocytochemistry. Important hepatic functions, including LDL metabolic ability and urea production, were increased in iHep cells on day 10.ConclusionWe successfully induced cBMSCs to differentiate into functional iHep cells. To our knowledge, this is the first report of canine liver tissue differentiation using Foxa1 and Hnf4α gene transfection. Canine iHep cells are expected to provide insights for the construction of liver models for drug discovery research and may serve as potential therapeutics for canine liver disease.

ATRA induces the differentiation of hepatic progenitor cells by upregulating microRNA-200a.

Hepatic progenitor cells (HPCs) are potential seed cells for hepatocyte transplantation treatment of liver diseases. ATRA can induce the differentiation and mature function of hepatic progenitor cells, but the mechanism is still poorly understood. Here, by using microRNA array to analyze the expression profiles of microRNA (miR), we found that miR-200 family molecules in HPCs were upregulated after ATRA treatment, especially miR-200a-3p, 200c-3p, and 141-3p. ATRA induction could downregulate the expression of hepatic stem markers Oct4 and AFP, and improve the expression of hepatic markers ALB, CK18, and TAT, and the activity of ALB-GLuc, as well as indocyanine green uptake and glycogen storage function of HPCs. These above effects of ATRA on HPC differentiation were almost inhibited by blocking of miR-200a-3p, but not miR-200c-3p and 141-3p using antagomir. Cell autophagy is associated with ATRA regulation in HPCs, compared with control group, the expression of LC3 and Beclin1 increased in ATRA-treated HPCs, and orange and red fluorescent spot, which represents autophagy flow, also enhanced after ATRA treatment. However, ATRA-induced cell autophagy level was inhibited in antagomir-200a-3p+ATRA-treated cells. Therefore, the present study indicates that antagomir-200a-3p is related to ATRA-induced hepatic differentiation of HPCs through regulating cell autophagy, supporting the possible use of ATRA as a key inducer in HPC-based therapy of liver diseases.

Blocking development of liver fibrosis augments hepatic progenitor cell-derived liver regeneration in a mouse chronic liver injury model.

The roles of hepatic progenitor cells (HPCs) in regeneration of a diseased liver are unclear. Hepatic stellate cells (HSCs) contribute to liver fibrosis but are also a component of the HPC niche. Hepatic progenitor cells expand along with HSC activation and liver fibrosis. However, little is known about the interplay of liver fibrosis and HPC-mediated liver regeneration. This study aimed to investigate HSCs and HPCs in liver regeneration. Liver injury in mice was induced with 3,5-diethoxycarbonyl-1,4-dihydrocollidine, and HPC expansion and fibrosis were assessed. An angiotensin II type 1 receptor blocker (ARB) was administered to assess its effect on fibrosis and regeneration. Treatment with ARB attenuated fibrosis and expansion of α-smooth muscle actin-positive activated HSCs as indicated by increased liver weight and Ki-67-positive hepatocytes. Immunohistochemical staining suggested that HPC differentiation was shifted toward hepatocytes (HCs) when ARB treatment decreased HPC encapsulation by HSCs and extracellular matrix. Conditioned medium produced by culturing the human HSC LX-2 line strongly augmented differentiation to biliary epithelial cells (BECs) but inhibited that to HCs. Activated HSCs expressed Jagged1, a NOTCH ligand, which plays a central role in differentiation of HPCs toward BECs. Hepatic stellate cells, the HPC niche cells, control differentiation of HPCs, directing them toward BECs rather than HCs in a diseased liver model. Antifibrosis treatment with an ARB preferentially redirects HPC differentiation toward HCs by blocking the NOTCH pathway in the HPC niche, resulting in more efficient HPC-mediated liver regeneration.

Regulation of Long Non-Coding RNA-Dreh Involved in Proliferation and Migration of Hepatic Progenitor Cells during Liver Regeneration in Rats.

Liver regeneration plays a significant role in protecting liver function after liver injury or chronic liver disease. Long non-coding RNAs (lncRNAs) are considered to be involved in the proliferation of hepatocytes and liver regeneration. Therefore, this study aimed to explore the effects of LncRNA-Dreh on the regulation of hepatic progenitor cells (HPCs) during liver regeneration in rats. Initially, the rat model of liver injury was established to investigate the effect of LncRNA-Dreh down-regulation on liver tissues of rats with liver injury. Subsequently, HPCs line WB-F344 cells were transfected with interference plasmid of LncRNA-Dreh and the expression of LncRNA-Dreh and Vimentin was detected. The proliferation and migration ability of WB-F344 cells, as well as the content of albumin (ALB) and alpha fetoprotein (AFP) in cell differentiation were then determined. Disorderly arranged structure of liver tissue, a large number of HPCs set portal area as center extended to hepatic lobule and ductular reaction were observed in liver tissues of rats with liver injury. The expression of LncRNA-Dreh decreased while Vimentin increased in liver tissues of rats with liver injury. Moreover, the proliferation and migration ability, expression of Vimentin and AFP in WB-F344 cells were increased after silencing of LncRNA-Dreh and ALB was decreased. Collectively, our findings demonstrate that inhibition of LncRNA-Dreh can enhance the proliferation and migration abilities of HPCs in liver regeneration but cause abnormal differentiation of HPCs.

Lipopolysaccharide induces the differentiation of hepatic progenitor cells into myofibroblasts constitutes the hepatocarcinogenesis-associated microenvironment.

Hepatocellular carcinoma (HCC) generally occurs in the presence of chronic liver injury, often as a sequela of liver fibrosis. Hepatic progenitor cells (HPCs) are known to be capable of forming both hepatocytes and cholangiocytes in chronic liver injury, which are also considered a source of myofibroblasts and tumor-initiating cells, under carcinogenic circumstances. However, the underlying mechanisms that activate HPCs to give rise to HCC are still unclear. In current study, the correlation between HPCs activation and liver fibrosis and carcinogenesis was investigated in rats and human specimens. We analyzed the role of HPCs in tumorigenesis, by transplanting exogenous HPCs in a diethylnitrosamine-induced rat HCC model. Our data indicated that HPC activation correlated with hepatic fibrosis and hepatocarcinogenesis. We further found that exogenous HPC infusion promoted liver fibrosis and hepatocarcinogenesis, while lipopolysaccharides (LPS) played an important role in this process. However, results of our study indicated that LPS did not induce HPCs to form tumor in nude mice directly. Rather, LPS induced myofibroblast-like morphology in HPCs, which enhanced the tumorigenic potential of HPCs. Further experiments showed that LPS/Toll-like receptor 4 (TLR4) signaling mediated the differentiation of HPCs into myofibroblasts and enhanced the production of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), which led to the aberrant expression of Ras and p53 signaling pathways in HPCs, and finally, promoted the proliferation and malignant transformation of HPCs, by long non-coding RNA regulation. Besides, examination of HCC clinical samples demonstrated that IL-6 and TNF-α production correlated with HPC activation, hepatic fibrosis, and HCC recurrence. Our study indicates that both myofibroblasts and tumor cells are derived from HPCs. HPC-derived myofibroblasts create tumor microenvironment and contribute to the proliferation and malignant transformation of HPCs. Furthermore, LPS present in the chronic liver inflammation microenvironment might play an important role in hepatocarcinogenesis, by regulating the plastic potential of HPCs.

THU-371-Modulation of H3K27 methylation by EZH2 and UTX/JMJD3 small molecules inhibitors impacts on proliferation and differentiation of hepatic progenitor cells and differentiated hepatocytes plasticity

THU-371-Modulation of H3K27 methylation by EZH2 and UTX/JMJD3 small molecules inhibitors impacts on proliferation and differentiation of hepatic progenitor cells and differentiated hepatocytes plasticity

Autophagy promotes hepatic differentiation of hepatic progenitor cells by regulating the Wnt/\u03b2-catenin signaling pathway

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.

ERRATUM: Gypenosides Ameliorate Carbon Tetrachloride-Induced Liver Fibrosis by Inhibiting the Differentiation of Hepatic Progenitor Cells into Myofibroblasts.

ERRATUM: Gypenosides Ameliorate Carbon Tetrachloride-Induced Liver Fibrosis by Inhibiting the Differentiation of Hepatic Progenitor Cells into Myofibroblasts.

Identification of a Novel Bone Marrow Cell-Derived Accelerator of Fibrotic Liver Regeneration Through Mobilization of Hepatic Progenitor Cells in Mice.

Granulocyte colony stimulating factor (G-CSF) has been reported to ameliorate impaired liver function in patients with advanced liver diseases through mobilization and proliferation of hepatic progenitor cells (HPCs). However, the underlying mechanisms remain unknown. We previously showed that G-CSF treatment increased the number of bone marrow (BM)-derived cells migrating to the fibrotic liver following repeated carbon tetrachloride (CCl4 ) injections into mice. In this study, we identified opioid growth factor receptor-like 1 (OGFRL1) as a novel BM cell-derived accelerator of fibrotic liver regeneration in response to G-CSF treatment. Endogenous Ogfrl1 was highly expressed in the hematopoietic organs such as the BM and spleen, whereas the liver contained a relatively small amount of Ogfrl1 mRNA. Among the peripheral blood cells, monocytes were the major sources of OGFRL1. Endogenous Ogfrl1 expression in both the peripheral blood monocytes and the liver was decreased following repeated CCl4 injections. An intrasplenic injection of cells overexpressing OGFRL1 into CCl4 -treated fibrotic mice increased the number of HPC and stimulated proliferation of hepatic parenchymal cells after partial resection of the fibrotic liver. Furthermore, overexpression of OGFRL1 in cultured HPC accelerated their differentiation as estimated by increased expression of liver-specific genes such as hepatocyte nuclear factor 4α, cytochrome P450, and fatty acid binding protein 1, although it did not affect the colony forming ability of HPC. These results indicate a critical role of OGFRL1 in the mobilization and differentiation of HPC in the fibrotic liver, and administration of OGFRL1-expressing cells may serve as a potential regenerative therapy for advanced liver fibrosis. Stem Cells 2019;37:89-101.

Hepatic stellate cells regulate hepatic progenitor cells differentiation via the TGF‐β1/Jagged1 signaling axis

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.