Differentiation of putative hepatic stem cells derived from adult rats into mature hepatocytes in the presence of epidermal growth factor and hepatocyte growth factor

Oval cells, putative hepatic stem cells, can differentiate into a wide range of cell types including hepatocytes, bile epithelial cells, pancreatic cells and intestinal epithelial cells. In this study, we used different growth factor combinations to induce oval cells to differentiate into mature hepatocytes. We isolated and purified oval cells utilizing selective enzymatic digestion and density gradient centrifugation. Oval cells were identified by their morphological characteristics and the strong expressions of OV-6, albumin, cytokeratin (CK)-19 and CK-7. Using a 2-step induction protocol, we demonstrated that oval cells first changed into small hepatocytes, then differentiated into mature hepatocytes. Small hepatocytes were distinguished from oval cells by their morphological features (e.g. round shape and nuclei) and the lack of CK-19 mRNA expression. Mature hepatocytes were identified by their ultrastructural traits and their expressions of albumin, CK-18, tyrosine aminotransferase (TAT), and alpha-1-antitrypsin (alpha-1-AT). Differentiated cells acquired the functional attributes of hepatocytes in that they secreted albumin and synthesized urea at a high level throughout differentiation. Oval cells can thus differentiate into cells with the morphological, phenotypic and functional characteristics of hepatocytes. This 2-step induction procedure could provide an abundant source of hepatocytes for cell transplantation and tissue engineering.     

Bronchioalveolar morphogenesis of human bronchial epithelial cells depending upon hepatocyte growth factor

Lung alveolar regeneration occurs in adult human lungs as a result of proliferation, differentiation and alveolar morphogenesis of stem cells. It is increasingly being believed that bronchial epithelial cells (BECs) have a potential as stem cells, because they are potent to differentiate into multiple central and peripheral lung cell types in three‐dimensional (3D) cultures, and they develop multiple foci with well‐differentiated histogenesis after transformed into neoplastic cells. In this study, we investigated morphogenic abilities of HBE135 human BECs immortalized by E6/E7 oncogene in 3D cultures. When HBE135 cells were cultured alone or co‐cultured with endothelial cells, the cells formed spherical colonies without branching. However, in co‐culture with lung fibroblast MRC‐9 cells, HBE135 cells formed colonies with bronchioalveolar‐like complex branching, suggesting that MRC‐9‐derived soluble factor(s) are responsible for the branching formation. MRC‐9 cells, not endothelial cells, were found to highly express hepatocyte growth factor (HGF), a soluble molecule involved in liver and kidney regeneration. An anti‐HGF neutralizing antibody severely suppressed the complex branching formation, but addition of HGF could not sufficiently compensate the morphogenic effects of MRC‐9 cells, suggesting that MCR‐9‐derived HGF was necessary but insufficient for the bronchioalveolar structure formation. Immunohistochemistry revealed that Met, a cognate receptor for HGF, was highly expressed and phosphorylated in neoplastic BECs from lung adenocarcinomas with well‐differentiated, not poorly differentiated, histogenesis. These results are consistent with the notion that BECs have an aspect of stem cells. This aspect appears to become manifest through HGF–Met signalling pathway activation.     

Effect of scatter factor and hepatocyte growth factor on motility and morphology of mdck cells

Summary We investigated the effects of human placental scatter factor (hSF), mouse scatter factor (mSF) and recombinant human hepatocyte growth factor (HGF) on motility and morphology of individual Madin-Darby canine kidney cells using a computerized cell tracking system. All three factors increased the velocity of individual cells and the ratio of moving to stationary cells. Similarly, all three factors caused changes in morphologic features of cells, leading to increased area, flatness, and polarity. Increases in area and flatness but not polarity were slightly greater with HGF than with hSF or mSF. These results suggest that SFs and HGF have similar effects on motility and morphology of isolated epithelial cells.     

Effect of hepatocyte growth factor/scatter factor and other growth factors on motility and morphology of non-tumorigenic and tumor cells

Summary Using an automated cell analyzer system, the effect of hepatocyte growth factor/scatter factor (HGF/SF), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), endothelial acidic fibroblast growth factor (a-FGF), platelet derived growth factor (PDGF), and recombinant human insulinlike growth factor (IGF) on the motility and morphology of Madin-Darby canine kidney (MDCK), rat hepatomas, C₂, and H_5–6 and murine mammary carcinoma (EMT-6) cells was investigated. Treatment of MDCK cells with HGF/SF, bFGF, EGF, and a-FGF resulted in an increase in average cell velocity and in the fraction of moving cells. Cells treated with the PDGF and IGF did not show significant alterations in velocity. MDCK cells treated with each growth factor were classified into groups of “fast” and “slow” moving cells based on their average velocities, and the average morphologic features of the two groups were quantitated. Fast-moving cells had larger average area, circularity, and flatness as compared to slow-moving cells. Factors that stimulated cell movement also induced alterations in cell morphologic parameters including spreading, flatness, area, and circularity. HGF/SF also scattered and stimulated motility of C₂ and H_5–6 hepatoma cells. In contrast to MDCK cells, there was no significant difference between the morphology of the fast moving and slow moving C₂ and H_5–6 cells. These studies suggest that growth factor cytokines have specific effects on motility of normal and tumor cells.     

Effects of some antibiotics on the growth of human diploid skin fibroblasts in cell culture

Summary During serial subcultures 50 μg per ml gentamicin and penicillin (100 U per ml)-streptomycin (100 μg per ml) depressed cell growth significantly 2 weeks after the addition of the antibiotics; gentamicin, but not penicillin-streptomycin, stimulated cell growth before it became inhibitory. Removal of the antibiotics resulted in the cell yield returning to normal. The results show that these antibiotics can be harmful to cells even at concentrations thought to be safe.     

Apoptosis of serum-free C2.8 mouse embryo hepatocytic cells caused by hepatocyte growth factor deprivation

C2.8 mouse embryo hepatocytic cells, acutely required exogenous hepatocyte growth factor (HGF) to survive and proliferate in serum-free Dulbecco's modified Eagle's medium supplemented with insulin, transferrin and Na-selenite. Greater than 90% of cultured C2.8 cells died within 48 hours from plating in the absence of HGF. Conversely, HGF prolonged maintenance of life and stimulated cell proliferation. Removal of HGF from the medium of cultures that had grown to confluency, also resulted in a rapid decreased cell survival. In the last circumstance, light microscopic observations revealed, with high frequency, morphological features characteristic of apoptosis. DNA within the affected cells underwent rapid fragmentation, revealed as a ladder of DNA fragments in multiples of about 200 base pairs. HGF prevented loss of cell viability, morphological damages and retarded DNA fragmentation in confluent C2.8 cells. Cycloheximide delayed cell death caused by HGF deprivation.     

Mechanism of TNF‐α‐induced migration and hepatocyte growth factor production in human mesenchymal stem cells

Accumulating evidence suggests that mesenchymal stem cells (MSCs) may decrease destructive inflammation and reduce tissue loss. Tumor necrosis factor‐α (TNF‐α) plays a central role in induction of proinflammatory signaling and paradoxically activates intracellular anti‐inflammatory survival pathways. In this study, we investigated whether TNF‐α could induce a chemotactic effect on human MSCs and stimulate their production of anti‐inflammatory factors in vitro, as well as determined mechanisms that mediated this effect. Migration assays demonstrated that TNF‐α had a chemotactic effect on MSCs. TNF‐α increased both hepatocyte growth factor (HGF) mRNA expression in MSCs and HGF secretion in conditioned medium. These effects were dependent on the p38 MAPK and PI3K/Akt, but not JNK and ERK signaling pathways. Furthermore, these effects were inhibited by a specific neutralizing antibody to TNF receptor II, but not TNF receptor I. We conclude that TNF‐α can enhance human MSCs migration and stimulate their production of HGF. These effects are mediated via a specific TNF receptor and signaling pathways. J. Cell. Biochem. 111: 469–475, 2010. © 2010 Wiley‐Liss, Inc.     

Met identification on human platelets: role of hepatocyte growth factor in the modulation of platelet activation

Circulating HGF is significantly increased in a number of thrombus-associated disorders. Since platelets play a pivotal role in thrombogenesis, the ability of HGF to interact with human platelets was investigated. This paper shows for the first time that human platelets express HGF receptor, the tyrosine kinase encoded by c-MET gene. At physiological concentrations HGF was found to inhibit both glycoprotein (alpha)IIb(beta)3 activation and thrombin-dependent platelet aggregation in a dose- and time-dependent manner. These results suggest that circulating HGF may counteract thrombogenesis by negatively modulating platelet functions.     

High expression of Met/hepatocyte growth factor receptor suppresses tumorigenicity in NCI-H1264 lung carcinoma cells.

The protein product of c-met proto-oncogene, Met, is a tyrosine kinase receptor for the hepatocyte growth factor (HGF). Met receptor is expressed in normal human bronchial epithelium. In comparison, its expression in squamous cell carcinoma (SQCC) of the lung is markedly decreased in a great majority of cases. To understand further the role of Met receptor overexpression in non-small-cell lung carcinoma, we forced-expressed the full-length met cDNA in the NCI-H1264 (H1264) lung carcinoma cell line with low constitutive expression of this receptor. In vitro studies demonstrated that increased Met expression in H1264 cells resulted in strong inhibition of their ability to form soft agar colonies and in marked suppression of tumorigenicity in the subcutaneous tissue of immune-deficient mice. This is despite inconsistent alteration in the proliferation rate on plastic surfaces. Tumor cells explanted from occasional xenograft tumors formed by the Met-overexpressing H1264 cells also demonstrated marked down-regulation of the receptor protein levels as compared to the transplanted cells. The results suggest that constitutive overexpression of Met receptor may negatively regulate the malignancy of certain human lung cancer cells.     

Synergistic induction of hepatocyte growth factor in human skin fibroblasts by the inflammatory cytokines interleukin-1 and interferon-gamma

Hepatocyte growth factor (HGF) is one of the vital factors for wound healing. HGF expression markedly increases in wounded skin and is mainly localized in dermal fibroblasts. HGF expression level in human dermal fibroblasts in vitro, however, is low and thus may be stimulated by some factors in the process of wound healing. Candidates of the factors are inflammatory cytokines released by polymorphonuclear and mononuclear cells infiltrating the wounded area, but HGF production in human dermal fibroblasts is only slightly induced by interleukin (IL)-1, tumor necrosis factor (TNF)-alpha or interferon (IFN)-gamma. We here report that a combination of IL-1beta and IFN-gamma or a combination of TNF-alpha and IFN-gamma very markedly induced HGF production. The synergistic effect of the former was more marked than that of the latter. Synergistic effects of IL-1beta and IFN-gamma were observed at more than 10 pg/ml and 10 IU/ml, respectively, and were detectable as early as 12 h after addition. Neither IFN-alpha nor IFN-beta was able to replace IFN-gamma. HGF mRNA expression was also synergistically upregulated by IL-1beta and IFN-gamma. IL-1beta plus IFN-gamma-induced synergistic production of HGF was potently inhibited by treatment of cells with the extracellular signal-regulated kinase (ERK) kinase inhibitor PD98059 and the p38 inhibitor SB203580 but not by the c-Jun N-terminal kinase (JNK) inhibitor SP600125. Taken together, our results indicate that a combination of IL-1beta and IFN-gamma synergistically induced HGF production in human dermal fibroblasts and suggest that activation of ERK and p38 but not of JNK is involved in the synergistic effect.     

Expression of epidermal growth factor receptor and associated glycoprotein on cultured human brain tumor cells

The expression of epidermal growth factor (EGF-R) in normal glial and glioma cells grown in culture was examined by using several independent assays. Immunoprecipitation with the monoclonal antibody R1 of extracts from metabolically labeled glial and glioma cells revealed a protein of Mr approximately 170,000, with a migration in sodium dodecyl sulfate-polyacrylamide gels identical to the EGR-R of A431 epidermal carcinoma cells. Furthermore, in the majority of glioma extracts, a protein of Mr approximately 190,000 was specifically immunoprecipitated by this antibody. Similar results were obtained by immunoblotting with a second antibody directed against a synthetic peptide in the sequence of the v-erb-B oncogene. In cell lines expressing both proteins, each was specifically phosphorylated on tyrosine in immune complex kinase assays. The majority of glioma cells bound between 40,000 to 80,000 125I-labeled epidermal growth factor molecules per cell. These results suggest that the expression of EGF-R is common in cultured human glioma cells. In addition, a structurally related protein, is expressed in some of these cells.     

人肝细胞生长因子基因表达质粒的构建及其活性研究

目的 :构建一种携带人肝细胞生长因子 (HGF)基因的表达质粒 (pUDKH) ,并对其体外活性进行研究 ,为其体内应用提供依据。方法 :从人胎盘cDNA文库用PCR方法克隆人HGF基因 ,并将其克隆至自行构建的真核表达载体 pUDK上 ,获得表达质粒 pUDKH。将pUDKH体外转染原代培养的骨骼肌细胞 ,分析其转染效率及表达上清中HGF、血管内皮生长因子 (VEGF)的表达水平 ,并采用MTT法分析不同剂量HGF表达产物对人脐静脉内皮细胞的作用。结果 :所克隆构建的携带人HGF基因的质粒 pUDKH可有效转染原代培养的骨骼肌细胞 (0 .0 5 7% ) ,并表达HGF(16～ 18ng/ 4× 10 5cells)和VEGF ,其表达产物对人脐静脉内皮细胞具有明显的增殖刺激活性 (P <0 .0 5 ) ,而且有剂量效应关系。结论 :初步证实本研究构建的质粒 pUDKH具有体内治疗缺血性疾病的应用潜力     

Insulin-like growth factor 1 (IGF-I) improves hepatic differentiation of human bone marrow-derived mesenchymal stem cells

The ability of MSCs (mesenchymal stem cells) to differentiate between other cell types makes these cells an attractive therapeutic tool for cell transplantation. This project was designed to improve transdifferentiation of human MSCs into liver cells using IGF-I (insulin-like growth factor 1) which, despite its important role in liver development, has not been used for in vitro hepatic differentiation. In the present study, the MSCs derived from healthy human bone marrow samples were cultured and characterized by immunophenotyping and differentiation potential into osteoblast and adipocytes. Transdifferentiation into hepatocyte-like cells was performed in the presence/absence of IGF-I in combination with predefined hepatic differentiation cocktail. To evaluate transdifferentiation, morphological features, immuno-cytochemical staining of specific biological markers and hepatic functions were assessed. Morphological assessment and evaluation of glycogen content, albumin and AFP (α-feto protein) expression as well as albumin and urea secretion revealed statistically significant difference between experimental groups compared with the control. Morphology and function (albumin secretion) of IGF-I-treated cells were significantly better than IGF-I-free experimental group. To the best of our knowledge, our study is the first to demonstrate that the combination of IGF-I with the predefined hepatic differentiation cocktail will significantly improve the morphological features of the differentiated cells and albumin secretion.     

Effects of hepatocyte growth factor overexpressed bone marrow‐derived mesenchymal stem cells on prevention from left ventricular remodelling and functional improvement in infarcted rat hearts

Bone marrow‐derived mesenchymal stem cells (BM‐MSCs ) transplantation has been reported to be a promising therapy for myocardial infarction (MI). However, low survival rate of BM‐MSCs in infarcted heart is one of the major limitations for the perspective clinical application. In this study, we aimed to investigate the effect of hepatocyte growth factor (HGF) on left ventricular function improvement of HGF gene‐modified BM‐MSCs (HGF‐MSCs) after its delivery into the infarcted rat hearts. BM‐MSCs were isolated with fibroblast‐like morphology and expressed CD44+CD29+CD90+/CD34‐CD45‐CD31‐CD11a. After 5‐azacytidine induction in vitro, 20%–30% of the cells were positively stained for desmin, cardiac‐specific cardiac troponin I and connexin‐43. Histological staining revealed that 2 weeks after MI is an optimal time point with decreased neutrophil infiltration and increased vascular number. Minimal infarct size and best haemodynamic analysis were also observed after cell injection at 2 weeks compared with that of 1 h, 1 week or 4 weeks. Echocardiogram confirmed that transplantation with HGF‐MSCs significantly improved left ventricular function compared with other groups in rat MI models. MSCs and HGF‐MSCslabelled with DAPI were detected 4 weeks after MI in the infarcted area. Decreased infarcted scar area and increased angiogenesis formation could be found in HGF‐MSCs group than in other groups as demonstrated by hematoxylin and eosin (H&E) staining and factor VIII staining. These results indicate that HGF‐MSCs transplantation could enhance the contractile function and attenuate left ventricular remodelling efficiently in rats with MI. Copyright © 2012 John Wiley & Sons, Ltd.     

肝细胞生长因子对人羊水干细胞迁移和黏附能力的影响

目的分离培养及鉴定羊水干细胞（hAFSC）,并研究肝细胞生长因子（HGF）对羊水干细胞迁移、黏附能力的影响。方法使用细胞贴壁法分离培养羊水干细胞,细胞免疫荧光及westernblot鉴定羊水干细胞,Transwell小室分析HGF对羊水干细胞迁移的作用。明胶贴壁法分析HGF对羊水干细胞黏附能力的作用。两组之间数据的比较采用独立样本t检验。结果分离的羊水干细胞均表达特异性标记物Oct-4、c-kit、SSEA-4、CD105。HGF在体外对hAFSC的迁移有趋化作用,对照组和HGF组每个视野的迁移细胞数分别为38±2.5和80±3.2。对黏附能力有促进作用,对照组和HGF组每个视野的黏附细胞数分别为19±1.5和50±2.7,差异均有统计学意义（P〈0.01）。结论 HGF可趋化羊水干细胞的迁移,增强羊水干细胞的黏附能力。     

Growth and differentiation in cultured human thyroid cells: Effects of epidermal growth factor and thyrotropin

Summary Human thyroid cells were grown and subcultured in vitro to examine their responses to known hormones and growth factors, and to serum. The cells were obtained from surgical specimens and were either neoplastic or nonneoplastic. The effects of culture conditions on cell growth were measured by changes in cell numbers and by stimulation of [³H]thymidine incorporation. The results showed that serum (0.5%) was essential for cell proliferation, and that a mixture of insulin (10 μg/ml), transferrin (5 μg/ml), hydrocortisone (10 μg/ml), somatostatin (10 ng/ml), and glycyl-histidyl-lysine (10 ng/ml) enhanced the effect of serum. Maximum growth of the cells was obtained when epidermal growth factor was present at 10⁻⁹ M. Differentiation was measured by production of thyroglobulin, which was found to be stimulated by thyrotropin. This system provides a means to study the hormonal control of growth and differentiation in human thyroid cells. This work was supported by grants from the Medical Research Council of Canada; the Department of Medicine, University of Toronto; and the National Cancer Institute of Canada. J. E. E. is a C.H. Best Foundation and Department of Medicine postdoctoral fellow.