Tuesday, May 17, 2022

Human Hepatic Stellate Cell Line

Small Interfering Rna Transfection

Protocol described for obtaining hepatic stellate cells from stem cells

To examine the influence of CHOP expression in LX2 cells treated with GGA, we performed experiments using siRNA transfection. RNAi was obtained from Dharmacon and transfected to LX2 cells according to the manufacturers protocol. Cells were used for experiments 24 h after transfection. To confirm knockdown of CHOP, RT-PCR was performed as described above.

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Activated Hepatic Stellate Cells Within The Hepatic Tumor Microenvironment

The interplay between liver tumor cells and the hepatic TME is crucial to the initiation and progression of HCC . The TME is defined as a peritumoral space contributing to the acquisition of various hallmark traits of cancer, including sustained proliferative signaling and activation of invasion, metastasis, and angiogenesis . Furthermore, the TME can be divided into two major components: cellular and non-cellular. Activated HSCs are a part of the cellular component and exhibit essential biological functions such as promotion of fibrogenesis and ECM remodeling to positively influence HCC tumorigenesis .

In addition to HSCs, cellular components of the hepatic TME include stromal hepatocytes, immune cells such as myeloid-derived suppressor cells , tumor associated macrophages , and cancer associated fibroblasts . Non-cellular components include cytokines such as Interleukin-6 and Interleukin-22 , growth factors such as VEGF , TGFB , PDGF , and CCN2 . Additional non-cellular components include matrix metalloproteinases , their inhibitors and proteoglycans . The following studies will cover the different roles activated HSCs play in HCC progression through their interaction with the other cellular and non-cellular components of the hepatic TME.

Table 1. Components of the hepatic TME.

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Isolation Of Human Hscs Using Density Gradient Centrifugation

Friedman first successfully isolated, cultured, and characterized human HSCs from normal human livers. Isolation of human HSC was also reported in other studies using density gradient centrifugation method . In general, researchers isolated human HSCs from wedge sections of human liver unsuitable for transplantation within 48 h. Sections of donor liver were isolated by catheter perfusion , or finely minced , and digested using pronase and collagenase, followed by density gradient centrifugation using Larex or other gradient medium to remove other non-parenchymal cells. HSCs isolated with this method were reported to be highly viable and with purity of ~ 90% . In some studies, HSCs isolated from density gradient centrifugation were further enriched and purified by centrifugal elutriation .

Density gradient separation remains the most widely used approach for HSC isolation, but this method targets the buoyancy of vitamin A-rich HSCs. This could result in inefficiency on isolating activated HSCs. It has been demonstrated that upon liver injury, large number of HSCs were retrieved from higher density gradient layers in rat .

Activated Hsc Regulation Of Angiogenesis Within The Hepatic Tme

Human macrophages exposed to HCV promote hepatic stellate ...

The effects of activated HSCs on angiogenesis in HCC have also been investigated over the past decade. Angiogenesis within the TME is essential for tumor progression, metastasis and invasion . Zhu et al. identified Interleukin-8 as a contributing factor to angiogenesis in HCC. Interestingly, IL-8 was highly expressed in HCC stroma and was mainly derived from activated HSCs rather than from HCC cells. Furthermore, an IL-8 neutralizing antibody was demonstrated to suppress tumor angiogenesis in Hep3B cells treated with conditioned media from activated HSCs. The authors also demonstrated similar results in vivo through a chick embryo chorioallantoic membrane assay. Most recently, Lin et al. observed that activated HSCs are the primary source of secreted angiopoietin-1 in human HCC cells in vitro. This not only describes the promotion of HCC angiogenesis through activated HSCs and Ang-1 expression, but also opens the potential of Ang-1 as an anti-angiogenic therapeutic target in HCC . These findings identify angiogenic factors produced by activated HSCs in the hepatic TME to promote hepatocarcinogenesis.

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Cellular Crosstalk Between Activated Hscs And Cellular Components Of The Hepatic Tme

The hepatic TME consists of various immune cells to create an immunosuppressed environment in order to maintain HCC tumor growth . Activated HSCs contribute to this immunosuppressed environment by secreting cytokines which induce MDSC expansion . In an orthotopic liver tumor mouse model, activated HSCs significantly increased regulatory T cell and MDSC expression to benefit HCC growth in the spleen, bone marrow, and tumor tissues . Furthermore, activated HSCs secrete angiogenic growth factors to form new vasculature within the TME . These functions of activated HSCs create a link to the circulatory system for supplying nutrients to the tumor. Immune cells may also regulate activation of HSCs in vitro, demonstrated by Interleukin 20 activation of HSCs, resulting in upregulation of TGFB1 and type I collagen, and increased proliferation and migration of activated HSCs . The same study further indicated that these fibrogenic phenotypes could be attenuated with an anti-IL-20 receptor monoclonal antibody, proposing IL-20 as a significant activator of HSCs and fibrogenesis. Taken together, activated HSCs may have an important role in promoting an immunosuppressed and angiogenic hepatic TME to support aggressive HCC cell growth.

Analysis Of Retinoid Biology

Retinol uptake and processing by LX-1 and LX-2 cells

Following plating and overnight incubation, cells were treated with two different concentrations of retinol for 24 hours in the dark. For this purpose, a concentrated stock solution of retinol was diluted in a small volume of ethanol , added to the media with 300 M palmitic acid to aid esterification, and then vigorously mixed. Following incubation, media from triplicate cultures were removed and cells scraped into 2 ml of ice cold PBS. Samples were then stored at 20°C prior to analysis. Control cells were cultured in medium with ethanol vehicle.

HPLC analyses of retinol and retinyl esters

Retinol and retinyl esters were analysed by reversed phase high pressure liquid chromatography , as described by Yamada and colleagues, using a Waters 510 HPLC pump operating at a flow of 1.8 ml/min, an Ultrasphere C18 column , and a Waters 996 Photodiodearray detector. Retinol and retinyl esters were monitored at 325 nm and quantified using standard curves relating the known mass to mass ratio of retinol or retinyl esters to that of internal standard retinyl acetate. This HPLC method allows for detection and quantitation of different retinyl esters, including retinyl palmitate, oleate, stearate, linoleate, and myristate.

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Comparative Quantitative Real Time Rt

HSCs, LX-1 and LX-2 cells were plated in 60 mm diameter dishes and cultured to 70% confluence. Cells were serum starved and supplemented with 0.2% BSA for 48 hours prior to TGF-1 treatment . LX-1 cells were additionally supplemented with 0.5% FBS. Control cells were also serum starved. Total RNA was extracted with TRIzol reagent according to the manufacturers instructions. Briefly, cells were lysed with the reagent, chloroform was added, and cellular RNA was precipitated by isopropyl alcohol. After washing with 75% ethanol, the RNA pellet was dissolved in nuclease free water.

Total RNA was reverse transcribed to complementary DNA using the Reverse Transcription System . RNA in 7.7 l of nuclease free water was added to 2.5 l of 10× reverse transcriptase buffer, 10 l of 25 mM MgCl2, 2.5 l of 10 mM dNTP, 1.0 l of random primer, 0.5 l of RNase inhibitor, and 0.8 l of AMV reverse transcriptase in a total volume of 25 l. The reaction was performed for 10 minutes at 25°C , 60 minutes at 42°C , and five minutes at 95°C .

Establishment And Characterization Of An Immortalized Human Hepatic Stellate Cell Line For Applications In Co

Hepatic stellate cell (Everything Human Cells) ð¬ð?ï¸?ðºðâ

XiaoPing Pan1,2, Yini Wang1,2, XiaoPeng Yu1,2, JianZhou Li1,2, Ning Zhou1,2, WeiBo Du1,2, YanHong Zhang1,2, HongCui Cao1,2, DanHua Zhu1,2, Yu Chen1,2, LanJuan Li1,2

1. State Key Laboratory for the Diagnosis and Treatment of Infectious Diseases, First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, 310003, China.2. Collaborative Innovation Center for the Diagnosis and Treatment of Infectious Diseases, Hangzhou, China.

Corresponding author: LanJuan Li, M.D. Collaborative Innovation Center for the Diagnosis and Treatment of Infectious Diseases, State Key Laboratory for the Diagnosis and Treatment of Infectious Disease, First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, 310003, China. Tel: 86-571-87236759 Fax: 86-571 87236759 Email: ljliedu.cn.More

Citation:Int J Med Sci

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Genetic Characteristics Of The Human Hepatic Stellate Cell Line Lx

  • Affiliation Institute of Clinical Chemistry and Pathobiochemistry, RWTH Aachen University, Aachen, Germany

  • Contributed equally to this work with: Ralf Weiskirchen, Jörg Weimer

    Affiliation Department of Gynaecology and Obstetrics, UKSH Campus Kiel, Kiel, Germany

  • Affiliation Institute of Clinical Chemistry and Pathobiochemistry, RWTH Aachen University, Aachen, Germany

  • Affiliation Center for Human Genetics, Bioscientia, Ingelheim, Germany

  • Affiliation Center for Human Genetics, Bioscientia, Ingelheim, Germany

  • Affiliation Institute of Human Genetics, University Hospital Schleswig-Holstein & Christian-Albrechts University Kiel, Kiel, Germany

  • Affiliation Department of Gynaecology and Obstetrics, UKSH Campus Kiel, Kiel, Germany

  • Affiliation Institute of Human Genetics, University Hospital Schleswig-Holstein & Christian-Albrechts University Kiel, Kiel, Germany

  • Affiliation Institute of Liver Diseases, Shanghai University of Traditional Chinese Medicine Shuguang Hospital, Shanghai, PR China

  • Affiliation Division of Liver Diseases, Mount Sinai School of Medicine, New York, New York, United States of America

  • Affiliations Center for Human Genetics, Bioscientia, Ingelheim, Germany, Department of Human Genetics, RWTH Aachen University, Aachen, Germany, Center for Clinical Research, University Hospital Freiburg, Freiburg, Germany

Effects Of Siniruangan Recipe On Proliferation Apoptosis And Activation Of Human Hepatic Stellate Cell Line Lx

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Human Hepatic Stellate Cell Lines

There are obvious disadvantages in obtaining and usage of primary HSCs, particularly primary human HSCs, such as the heterogeneity of isolated cell populations and cellular characteristics, limited supply, considerable variations of cell preparation in different laboratories, as well as the isolation equipment and techniques requirements. Immortalized HSC lines were established and have been used in a wide range of research. These immortalized cell lines provide unlimited resource supply, homogeneity, and are suitable for genetic manipulation studies. They recapitulate many activated HSC features, and can serve as a useful tool for mechanistic investigation of HSC function in hepatic fibrosis and liver pathophysiological processes. The immortalized HSC lines currently in use have been generated from primary HSC through spontaneous immortalization during long-term culture, or by transformation with the simian virus 40 large T-antigen , or ectopic expression of human telomerase reverse transcriptase . Notably, none of the published cell lines are reported to be tumorigenic. Considering these cells are genetically modified, careful evaluation of the reported studies is always warranted .

Table 2 Characteristics of human hepatic stellate cell lines

Crosstalk Between Activated Hscs And Non

Human Hepatic Stellate Cell Line Lx 2

In addition to interacting with other hepatic TME cellular components, HSCs also respond to the non-cellular components of the liver TME . An example of such is the response to CCN2 produced from hepatic tumor cells . Makino et al. demonstrated that elevated CCN2 expression positively correlated with activated HSCs, indicated by smooth muscle actin expression, in both mouse and human liver tumors. Furthermore, the authors showed that anti-CCN2 reduced IL-6 production in LX-2 cells and inhibited STAT3 activation in HepG2 cells . This study was the first to establish HCC-cell-derived CCN2 activates HSCs in the TME, thus, accelerating the progression of HCC through cytokine production. These results also support the need for further exploration of CCN2 and other ECM proteins involved in the activation of HSCs.

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Human Hsc Isolation Methods

An efficient method of HSC isolation and clear characterization of human HSCs is undoubtedly critical for a deep understanding of its role in human liver physiology and liver diseases. Two main methods for isolating HSCs from human liver have been described so far, one is to grow smooth muscle-like cells from liver tissue explants, and the other is using density gradient centrifugation similar to the isolation of HSCs in rodents .

Hepatocyte Growth Factor And Transforming Growth Factor

To measure HGF and TGF-1 secretion in the HSC-Li cells, we inoculated 1.0 × 106 of HSC-Li cells into 90 mm dishes. After 24 h of culture, the cells were cultured with DMEM media supplemented with low amounts of FBS for eleven days. The culture supernatant of the HSC-Li cells was collected daily and assayed using human HGF and TGF-1 ELISA kit according to the manufacturer’s manual.

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Gga Decreased Density Of Hscs

We monitored LX2 cells treated with GGA using phase-contrast microscopy and observed a concentration-dependent decrease in cell density . Similarly, GGA reduced the density of HHSteCs in a concentration-dependent manner. In contrast, HepG2 cells showed no significant morphological or density changes with GGA treatment.

Fig. 1

Geranylgeranylacetone decreased LX2 and HHSteC density in a concentration-dependent manner. After treatment with GGA for 24 h, three different cell lines, LX2 cells, HHSteCs and HepG2 cells, were observed by phase-contrast microscopy. There were no significant morphological changes or cell density decrease in HepG2 cells

Determination Of Telomere Restriction Fragment Length

Tissue-engineered liver cells from universal donor cell line avoid immune response

The length of the telomere restriction fragment was determined by Southern blot analysis as described . Briefly, genomic DNA was isolated using the DNeasy kit and 2 g of the isolated genomic DNA digested with HinfI and RsaI, and fractionated in a 0.8% agarose gel. After transfer onto Magna Graph nylon transfer membrane , the filter was hybridized with an oligonucleotide 3, which was end-labeled with ATP. The filter was analyzed by autoradiography and by phosphorimager analysis . The maximal signal in each band was determined, and the length of the telomere restriction fragment calculated by comparison to that of a molecular size marker.

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Expression Of Intermediate Filaments

Cell lines were also examined by immunocytochemistry for expression of the intermediate filament proteins -SMA, vimentin, and GFAP, which are typically expressed in HSCs . Both LX-1 and LX-2 cells expressed -SMA, a marker for activated HSCs. Vimentin, a marker for cell types of mesenchymal origin, and GFAP, a marker of a subpopulation of stellate cells, were present in both cell lines in a cytoskeletal network. Western analysis also confirmed expression of these proteins .

Qualities Of Primary And Immortalized Hsc

Several features make HSC lines potentially appropriate for investigations analysing general cellular and biochemical aspects of HSC biology. They exhibit many HSC/MFB-specific features and express almost all relevant marker genes indicating that they originated from HSC. Moreover, comparison of individual lines with primary cells by microarray analysis revealed that the gene expression patterns are striking similar with expression of multiple neuronal and ECM genes, again suggesting that the cells originate from HSC . However, there are pronounced differences that are summarized in the following.

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General Aspects Of Immortal Hsc

Primary HSC provide characteristics very close to that observed in vivo, but have some significant disadvantages. First, there may be considerable variation in the cellular features of cells prepared in different laboratories or from different operators. In addition, there are a number of plausible reports describing the occurrence of heterogeneity within HSC cultures. Secondly, cells originating from one preparation have only limited utility because of their limited life span. Thirdly, sub-cultivation of primary HSC can be achieved only for a limited number of passages associated with modified characteristics, apoptosis, altered cytokine susceptibility and variations in gene expression . Fourthly, experiments requiring large numbers of cells or long-term growth are rarely possible. Moreover, there is considerable pressure on scientists to reduce or even eliminate the use of animals in research.

To overcome these limitations, many investigators have transferred established concepts to develop permanent HSC cell lines. Presently, immortal HSC cell lines were derived from primary HSC that were transformed with the simian virus 40 large T-antigen which has pleiotropic effects on cell division achieving this by binding to the transcription factor E2, p53âor pRB, manipulated by ectopic expression of telomerase reverse transcriptase activity, isolated from experimentally diseased livers, subjected to UV light or alternatively were spontaneously immortalized during culturing.

Nadph Oxidase Inhibition And Farnesoid X Receptor Activation Rescue Phh Functions In The Presence Of Activated Hscs

Human macrophages exposed to HCV promote hepatic stellate ...

In MPTCs , we observed an increase in the gene expression of NFE2L2 , a transcription factor involved in oxidative stress signaling . Reactive oxygen species generated via NOX1 and NOX4 enzymes can mediate fibrogenic pathways in HSCs and hepatocytes. We did not detect the expression of either NOX1 or NOX4 in MPCC controls, but both were detected in pure activated HSC cultures with NOX4 expression being higher than NOX1 . However, only NOX4 gene expression was detected in MPTCs, likely due to the contribution from the activated HSCs at the physiological seeding ratio. Since the inhibition of NOX1& 4 with the small molecule GKT137831 has shown anti-fibrotic effects in mouse models, we hypothesized that incubating MPTCs with GKT137831 could alleviate the hepatic dysfunctions observed due to the presence of activated HSCs. Furthermore, FXR is a major regulator of hepatocyte metabolism and bile production/transport that is downregulated in NASH patients. Activation of FXR via obeticholic acid has shown positive effects in ameliorating lipid accumulation and fibrosis in patients with NASH. Since we observed a strong downregulation of NR1H4 gene expression in PHHs, we hypothesized that treatment of MPTCs with OCA could alleviate hepatic dysfunctions. Finally, we tested the simultaneous effects of both drugs on MPTCs.

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