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ObjectiveTo investigate the effect of macrophages (MCs) on the differentiation of mouse induced pluripotent stem cells (iPSCs) into hepatic progenitor cells (HPCs). MethodsA total of 24 C57BL/6N 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. ResultsHPCs derived from iPSCs were established in vitro, and HPCs had the potential to differentiate into hepatocytes. Immunofluorescence assay showed that compared with the D12 control group, the D12 MC group had a significant increase in the protein expression of the HPC-specific protein CK19 (0.901±0.072 vs 0.686±0.097, t=-3.093, P＜0.05). Western blot showed that compared with the D12 control group, the D12 MC group had a significant increase in the protein expression of the HPC-related protein CK19 (1.922±0.103 vs 1.448±0.012, t =-7.881, P ＜005), as well as a significant increase in the protein expression of the autophagy-related protein LC3 (1.392±0.042 vs 1.101±0048, t =-5.978, P＜005). ConclusionMCs can promote the differentiation of mouse iPSCs into HPCs, possibly by increasing the autophagy level of HPCs.
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Ductular reaction is a kind of repair response to biliary and hepatocellular injury, and the pathological changes include lobular bile duct hyperplasia, matrix change and inflammatory cell infiltrations. In human liver, a ductular reaction originates from the activation and proliferation of hepatic progenitor cells. The activated hepatic progenitor cells, the intermediate hepatocytes and the reactive small bile duct cells together form the cell complex of ductular reaction, which has the dual characteristics of hepatobiliary system and co-expresses the hepatocyte and cholangiocyte antigen. Ductular reaction appears in all kinds of acute and chronic hepatobiliary injury, and plays an important role in liver regeneration and repair, fibrogenesis, cirrhosis formation, and hepatobiliary cancer occurrence and development. Understanding the pathological features and pathophysiological mechanisms of ductular reaction in various clinical hepatobiliary diseases and their role in liver regeneration can provide important suggestion for further exploring new-targeted treatment approach.
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AIM: To explore the influence of Huqi San on the Hedgehog signaling pathway in rats with prehe-patocarcinoma.METHODS: The model of prehepatocarcinoma in the rats was established by a modified solt-farber method.The rats were intragastric administrated with Huqi San solution for 3 d after subtotal hepatectomy.Four weeks after administration of the Huqi San solution, the hepatic damage was observed by histopathological analysis.The protein expression of glutathione S-transferase-π (GST-π), alpha-fetoprotein (AFP), OV6, albumin (ALB) and glioma-associated oncogene homolog 2 (Gli2) was detected by immunohistochemistry and immunofluorescence staining.The expression of Sonic hedgehog (Shh), Smoothened (Smo), Gli2, cyclin D and cyclin E at mRNA and protein levels in the rats was determined by RT-qPCR and Western blot, respectively.The serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and gamma-glutamyltransferase (GGT) were assayed using diagnostic kits.RESULTS: Compared with model group, Huqi San decreased the serum levels of ALT, AST and GGT, and alleviated the pathological changes in prehepatocarcinoma rats.Huqi San inhibited the protein expression of GST-π and AFP (P<0.05) in the prehepatocarcinoma rats.Huqi San also promoted the protein expression of OV6 and ALB (P<0.05).Furthermore, Huqi San activated Hedgehog signaling pathway and its downstream targeting molecules such as Shh, Smo, Gli2, cyclin D and cyclin E.In addition, the results in vitro showed that Huqi San may activate Hedgehog signaling pathway and promoted oval cell proliferation.CONCLUSION: Huqi San not only promotes hepatic progenitor cell proliferation, but also induces hepatic progenitor cell differentiation and inhibits prehepatocarcinoma in the rats probably via activating Hedgehog signaling pathway.
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Objective To isolate and cultivate mouse hepatic progenitor cells (mHPCs) from E14.5 mouse fetal liver in vitro and induce mHPCs differentiation into cholangiocytes.Methods Isolation of mHPCs from mouse fetal liver was based on the cell surface antigen delta-like protein 1/preadipocyte factor 1 (Dlk/Pref-1) by a fluorescence-activated cell sorter (FACS).Then mHPCs isolated were co-cultured with/without mouse embryonic fibroblasts (MEFs) by using Transwell.The cell antigen alpha-fetoprotein (AFP), albumin (ALB) and cytokeratin19 (CK19) expression in freshly isolated DLK1+cells or co-cultured for 4 days and 6 days were observed with immunocytochemical method.Results When co-cultured with MEFs, the division and proliferation were observed in most of DLK1+ cells and grape-like aggregation was formed.Cells began to adhere to growth and began to become spindle-shaped on 4th day.The DLK1+cells isolated freshly by FACS were expressed AFP and low levels of ALB but not expressed CK19.But, these cells expressed CK 19 and weak expression of ALB on 4th day.In addition, the expression of CK19 increased and the expression of ALB almost not detected on 6th day.Conclusions Most of DLK1+ cells, isolated from E14.5 fetal livers by FACS, are proved to be mHPCs.Furthermore, these cells can proliferate quickly and differentiate into cholangiocytes by co-culture with MEFs.
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The liver has a strong regenerative capacity,and in case of acute injury,the proliferation of mature hepatocytes helps to complete liver regeneration.However,in case of chronic injury,the proliferative capacity of mature hepatocytes is damaged or exhausted,and the activation,proliferation,and differentiation of hepatic progenitor cells are involved in liver regeneration.This article summarizes the characteristics and origins of hepatic progenitor cells,their role in tissue repair after liver injury and development of liver cancer,and potentials and problems of cell transplantation in the treatment of liver diseases.It is pointed out that an understanding of the biological characteristics of hepatic progenitor cells,their role in liver injury and liver cancer,and related pathogenesis helps with the treatment of liver diseases.
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Objective@#To investigate the regulatory effect of faciogenital dysplasia 6 (FGD6) gene on hepatic stem cell differentiation.@*Methods@#FGD6 gene was selected for the co-intervention of target sequence, the AdEasy system was used for the construction of adenovirus vector and the packaging and multiplication of the recombinant adenovirus vector pSES-FGD6-siRNA, and the HP14.5 cells were infected. Immunofluorescence assay was used to measure the expression of FGD6 protein in HP14.5 cells, quantitative real-time PCR was used to measure the mRNA expression of FGD6, alpha-fetoprotein (AFP), and albumin (Alb), and Western blot was used to measure the protein expression of FGD6, AFP, and Alb. The empty pSES-Ad-RFP adenovirus vector was constructed as control in each group. All data were expressed as x±s, and a one-way analysis of variance was performed.@*Results@#FGD6 protein was mainly expressed in the nucleus of HP14.5 cells. The pSES-FGD6-siRNA adenovirus vector was successfully constructed and it downregulated the expression of FGD6 gene and the mRNA and protein expression of AFP in HP14.5 cells and upregulated the mRNA and protein expression of Alb (P < 0.01).@*Conclusion@#The inhibition of the expression of FGD6 gene in HP14.5 cells may differentiate HP14.5 cells into hepatocytes. Therefore, FGD6 gene plays an important role in the differentiation regulation of hepatic stem cells.
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Objective:To explore the role of bone morphogenetic protein 4 (BMP-4) in hepatic progenitor cells (HPCs).Methods: The effect of BMP-4 on rat hepatic oval cells was examined by using the WB-F344 rat hepatocytic epithelial stem-cell-like cell line. This hepatocytic cell line could exert various hepatocyte functions including the secretion of albumin and urea. Immunohistochemistry was used to examine the effects of BMP-4 and its antagonist, Noggin, on the proliferation and differentiation of these cells, cellular uptake and excretion of indocyanine green, the periodic acid-schiff (PAS) assay for glycogen storage and the expression of hepatic markers.Results:Our results showed for the first time that BMP-4 may acted as a potential inducer of hepatic differentiation in rat hepatic oval cells.Conclusions:This cell source offers a much-needed attractive and expandable source for future investigations of drug screening, stem cell technologies and cellular transplantation, in a society with increasing levels of liver disease and damage.
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Objective: To explore the role of bone morphogenetic protein 4 (BMP-4) in hepatic progenitor cells (HPCs). Methods: The effect of BMP-4 on rat hepatic oval cells was examined by using the WB-F344 rat hepatocytic epithelial stem-cell-like cell line. This hepatocytic cell line could exert various hepatocyte functions including the secretion of albumin and urea. Immunohistochemistry was used to examine the effects of BMP-4 and its antagonist, Noggin, on the proliferation and differentiation of these cells, cellular uptake and excretion of indocyanine green, the periodic acid-schiff (PAS) assay for glycogen storage and the expression of hepatic markers. Results: Our results showed for the first time that BMP-4 may acted as a potential inducer of hepatic differentiation in rat hepatic oval cells. Conclusions: This cell source offers a much-needed attractive and expandable source for future investigations of drug screening, stem cell technologies and cellular transplantation, in a society with increasing levels of liver disease and damage.
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One of the most important characteristics of adult liver is its regenerative ability to maintain its original mass and function after injury. With remarkable self-replication ability, hepatocytes play a critical role in liver regeneration. When there is a restriction in the proliferation capacity and/ or a massive loss of mature hepatocytes,other cells in liver can contribute to liver regeneration in different ways. Deep study on the effects and mechanisms of various hepatic cells’contribution to liver regeneration is helpful for increasing the understanding about liver regeneration and providing new insights for diagnosis and management of various advanced liver diseases.
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OBJECTIVE@#To explore the role of bone morphogenetic protein 4 (BMP-4) in hepatic progenitor cells (HPCs).@*METHODS@#The effect of BMP-4 on rat hepatic oval cells was examined by using the WB-F344 rat hepatocytic epithelial stem-cell-like cell line. This hepatocytic cell line could exert various hepatocyte functions including the secretion of albumin and urea. Immunohistochemistry was used to examine the effects of BMP-4 and its antagonist, Noggin, on the proliferation and differentiation of these cells, cellular uptake and excretion of indocyanine green, the periodic acid-schiff (PAS) assay for glycogen storage and the expression of hepatic markers.@*RESULTS@#Our results showed for the first time that BMP-4 may acted as a potential inducer of hepatic differentiation in rat hepatic oval cells.@*CONCLUSIONS@#This cell source offers a much-needed attractive and expandable source for future investigations of drug screening, stem cell technologies and cellular transplantation, in a society with increasing levels of liver disease and damage.
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Hepatic progenitor cells (HPCs) are a potential cell source for liver cell transplantation but do not function like mature liver cells. We sought an effective and reliable method to induce HPC maturation. An immortalized HP14.5 albumin promoter-driven Gaussian luciferase (ALB-GLuc) cell line was established from HPCs isolated from fetal mouse liver of post coitus day 14.5 mice to investigate the effect of induction factors on ALB promoter. HP14.5 parental cells were cultured in DMEM with different combinations of 2% horse serum (HS), 0.1 µM dexamethasone (DEX), 10 ng/mL hepatic growth factor (HGF), and/or 20 ng/mL fibroblast growth factor 4 (FGF4). Trypan blue and crystal violet staining were used to assess cell proliferation with different induction conditions. Expression of hepatic markers was measured by semi-quantitative RT-PCR, Western blot, and immunofluorescence. Glycogen storage and metabolism were detected by periodic acid-Schiff and indocyanine green (ICG) staining. GLuc activity indicated ALB expression. The combination of 2% HS+0.1 µM Dex+10 ng/mL HGF+20 ng/mL FGF4 induced the highest ALB-GLuc activity. Cell proliferation decreased in 2% HS but increased by adding FGF4. Upon induction, and consistent with hepatocyte development, DLK, AFP, and CK19 expression decreased, while ALB, CK18, and UGT1A expression increased. The maturity markers tyrosine aminotransferase and apolipoprotein B were detected at days 3 and 6 post-induction, respectively. ICG uptake and glycogen synthesis were detectable at day 6 and increased over time. Therefore, we demonstrated that HPCs were induced to differentiate into functional mature hepatocytes in vitro, suggesting that factor-treated HPCs may be further explored as a means of liver cell transplantation.
Subject(s)
Animals , Mice , Cell Differentiation/drug effects , Embryo, Mammalian/drug effects , Hepatocytes/cytology , Liver/cytology , Stem Cells/drug effects , Antigens, Differentiation/analysis , Apolipoproteins B/isolation & purification , Cell Proliferation , Dexamethasone/administration & dosage , Fibroblast Growth Factors/administration & dosage , Gentian Violet , Glycogen/metabolism , Hepatocyte Growth Factor/administration & dosage , Indocyanine Green/pharmacokinetics , Primary Cell Culture/methods , Reverse Transcriptase Polymerase Chain Reaction , Stem Cells/cytology , Trypan Blue , Tyrosine Transaminase/isolation & purificationABSTRACT
Objective To explore the effect of recombinant adenovirus-mediated bone morphogenetic proteins 2, 7 (Adv- BMP2, Adv-BMP7) in inducing transformation of murine embryonic hepatic progenitor cells to mature hepatic-like cells. Methods HP14.5 cells were divided into 4 groups, and then infected by recombinant adenovirus expressing BMP2, BMP7, hepatocyte growth factor (HGF), and green fluorescent protein (GFP), respectively. For investigating the differential regulation of HP14.5 cells, the luciferase report gene was detected at the 1st, 4th and 7th day post infection, the expression of hepatocyte marker albumin (ALB) was detected at the 7th day after infection by cellular immunofluorescence assay. The maturation and differentiation of HP14.5 cells were examined by PAS staining and urea nitrogen synthesis of the cells at day 4, 7 and 10 post-infection. Results??The expression of ALB with BMP2 and HGF increased significantly compared to that in GFP control group tested by luciferase report gene; cellular immunofluorescence assay indicated that the specific marker of mature hepatocyte ALB was strong expressed at day 7 post-infection, while a negative result was observed in the GFP control group; HP14.5 cells infected with BMP2 and HGF have also acquired functional characteristics of hepatocytes which synthesized and secreted urea nitrogen, and stored glycogen. However, less inductive activity was found in BMP7 group. Conclusion BMP2 may induce the differentiation of HP14.5 cells into mature hepatocyte-like cells with initial synthesis and secretion, but BMP7 may have no such a capability.
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Objective To investigate the impact of wnt3 upon epithelial-mesenchymal transition in mouse hepatic progenitor cells (14-19 cells). Methods Ad-GFP-wnt3 virus and blank Ad-GFP were transfected into 14-19 cells; cell morphology was observed under microscope. The migration ability of 14-19 cells was examined by wound-healing assay and transwell assay. The expression of epithelial and mesenchymal markers in 14-19 cells was detected by real-time PCR and Western blotting analysis. Results Microscopic observation found that 14-19 cells changed from an epithelial characteristic shape into a spindle-like shape. Wound-healing assay and Transwell assay showed that the migration ability of 14-19 cells highly expressing wnt3 was significantly enhanced compared with the blank control group(P<0. 01). Real-time PCR and Western blotting analysis showed that mesenchymal markers N-cadherin, vimentin and twistl were significantly increased at both mRNA and protein levels in Ad-GFP-wnt3 virus group than the blank control group, while the epithelial marker E-cadherin was significantly decreased at mRNA and protein levels(P<0. 01). Conclusion Wnt3 can induce epithelial-mesenchymal transition in mouse hepatic progenitor cells, indicating that wnt3 may take part in the progress of hepatic fibrosis.
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Objective To observe the characteristics of hepatic progenitor cells(HPCs)activation in liver tissues of patients with hepatitis B cirrhosis,and to investigate the relationship between the number of HPCs and HBV infection.Methods Cytokeratin 7(CK7)-was stained immunohistochemically in liver tissues of 16 patients with hepatitis B cirrhosis.HPCs and duetular reactions were quantitively analyzed.The expression of HBsAg and HBcAg were also detected to evaluate its relationship with HPCs activation.Results HPCs were extensively activated and marked duetular reactions can be observed in cirrhotic liver tissues.Tlle expression of HBsAg was positively correlated with HPCs activation.Conclusions HPCs are extensively activated in cirrhotic liver tissues,and HBV infection may facilitate its activation.