Integrative Liver Cell

Since the last renewal submission, ILCC Cell Bank provided total 283 samples to 21 investigators from 17 institutions of which 18 are NIH-funded investigators. This service provides small aliquots of liver cells isolated from different ALD/fibrosis models to investigators who study or wish to study ALD but lack the resources and skills to isolate liver cells. Aliquots usually contain 0.5-8×106 primary cells/vial and 0.1-5×105 cells for MACS or FACS purified subclasses of the liver cells either as snap-frozen pellets or in RNA lysis buffer. This service is ideal for exploratory analysis. Once preliminary evidence is attained, investigators usually request full ILCC services to obtain samples in larger quantities. If larger quantity is required, we recommend the investigators to request frozen cells through the Cell Bank or freshly isolated cells from ILCC. The program will follow the same service priority for investigators to that describe above for the other ILCC services. For this service, ILCC works with Animal Core to isolate cells from different versions of mouse ALD models for a range of liver phenotype as described above.

Stephanie Pan is a primary core laboratory specialist responsible for isolation procedures, updating the ILCC request, recording activities, and organizing the Cell Bank services. She has independently been performing ILCC’s isolation procedures for the past 6 years. Steven Balog, Research Lab Supervisor, has been managing breeding colonies for the ILCC for the past 2.5 years and will continue to maintain commonly used genetic mice such as Col1a1-GFP, Rosa26mTmG mice, and 6 Cre mice (Lyz2Cre, Cx3cr1Cre, LratCre, AlbCre, CD133CreERT2, Col1a2CreERT2) required for cell-type specific genetic manipulations and lineage and cell fate analyses.

1. Due to the large volume of service requests, and our primary mechanism of funding, priority will be given to researchers with projects relevant to alcohol.
2. Requests for normal rat and mouse cells should be directed to Stephanie Pan (stephaqp@usc.edu or 323-442-3850) or Dr. Hidekazu Tsukamoto (htsukamo@med.usc.edu) at least 1-3 weeks prior to the time the cells are needed
3. Requests for cell isolation services from the disease models (as described under Animal Core Services), should be made to Stephanie Pan (stephaqp@usc.edu or 323-442-3850) or Hidekazu Tsukamoto (htsukamo@med.usc.edu) in advance.
4. The ILCC charges are based on a whole isolation procedure per rat or mouse and not on cell numbers isolated or attached for culture, because the cell yield and attachment vary.
5. Click here for tables summarizing the yield of cells, purity, and viability for commonly isolated cell types, the data which are based on 20 most recent preps performed (updated October 2022).
6. The services are rendered to Center or non-Center members on a charge back basis to recover the costs of supplies which are not supported by the Center and resource grants from NIAAA. See the chargeback fees in a table below for commonly performed services.
7. For preliminary trials and young investigators or trainees, subsidized fees may be considered. Consult Hidekazu Tsukamoto (htsukamo@med.usc.edu). 50% discount for “F” and “K” awardees.
8. Occasionally, the services can be rendered to those who are not included in the priority criteria, including industries, provided that the ILCC has the availability in its schedule.  Such services will have higher charge back rates, which include personnel costs and surcharges.
9. If you wish to know the techniques used to isolate cells, please click here.

The ILCC also provides or assists in specialized services requiring a combination of the disease model, genetic model, FACS isolation, cell labeling and transplantation and/or newly developed procedures by the ILCC as described below.

1. Genetic cell lineage and fate tracing:  The ILCC now performs cell lineage and cell tracing with collaboration with the Animal Core by using Rosa26mTmGflox reporter mice crossed with Alb-Cre (hepatocyte and biliary), Ttr-Cre (hepatocyte), Col1a1-Cre (activated HSC), Mesp1-Cre (mesenchyme), Wt1-Cre (mesothelium), Lysozyme-Cre (myeloid) to label respective cells for tracing in mouse models of ALD and liver fibrosis. Labeled cells are isolated by the Core via FACS using GFP and a fluorophore-conjugated antibody against a marker for the cell type of interest for biochemical characterization, culture experiments, or cell fate analysis via transplantation.
2. FACS isolation of activated HSC from Col1a1-GFP transgenic mice developing alcoholic liver fibrosis:  In the past 20 years, we have learned that isolation of pure activated HSCs from experimental alcoholic liver fibrosis is very challenging if not impossible. This is primarily because HSC become severely depleted of vitamin A as compared to activated HSC from other models of liver fibrosis induced by CCl4 or bile duct ligation, making it impossible to isolate them based on their normal light density. Fatty HC and HM which phagocytized lipids also contaminate the 1.035-1.045 density interface from which HSC are usually recovered. To overcome these problems, the ILCC now applies a FACS-based isolation technique to Col1a1-GFP mice to separate HSC based on their UV fluorescence for vitamin A content and the activation marker of type I collagen promoter activity as detected by GFP.  Using this method, a pure population of UV- or UV+ (mostly UV- but some UV+) and GFP+ cells is isolated as activated HSC as shown below in this figure.
3. CD133+ liver progenitor isolation:  As briefed above, the ILCC developed an isolation method of CD133+ liver progenitors. Livers from the mouse model of alcoholic hepatitis (1) are briefly perfused with 25mg/ml collagenase IV. CD45+ cells are depleted by MACS and CD45- cells are stained with CD133-APC, CD49f-PE, and CD45-eFlour 450 for FACS-based separation of CD133-CD49f+ cells. Note CD133+CD49f+ cell population is very low (<0.1%) in normal liver but can increase to >3% in AH liver. RNA-seq analysis of this population vs. CD133- cells, confirms CD133+ cells are indeed progenitors with higher expression of stem cell and progenitor markers. The presence of CD133+CD49f+ progenitor cells is only observed in this model of AH but not in chronic ASH, suggesting the former pathology uniquely induces progenitors.
4. Assessment of infiltrating vs. resident HM and tracking on blood monocytes: Xu, now serving as one of two Associate Directors for the ILCC, has succeeded in analyzing the relative contributions of resident HM vs. monocyte-derived HM to M1 macrophage activation in mouse ASH model. After isolation of peripheral blood monocytes, the cells are labeled with PKH26 fluorescent dye and injected intravenously into recipient ASH mouse (0.5×106 cells/mouse). Then, HM are isolated and further labeled with anti-CD45-V450, F4/80-FITC, and CX3CR1-APC antibodies. Using FACS, infiltrating donor monocyte-derived HM are separated as CD45+PHK26+F4/80+CX3CR1high cells, and recipient infiltrating monocyte-derived HM and recipient resident HM are separated as CD45+PHK26-F4/80+CX3CR1high and CD45+PHK26-F4/80+CXCR1low cells, respectively. This method allows investigating how these 2 populations contribute to inflammation in ASH and the role of a particular gene of interest in monocyte transmigration into ASH livers by using donor monocytes with ablation of the gene as used to demonstrate the important role of Notch1 (2).
5. Hypoxia system: As the liver is a relatively hypoxic organ and alcohol is known to exacerbate centrilobular hypoxia, the ability to perform cell culture studies under hypoxic conditions is important.  The ILCC utilizes a complete hypoxia system from BioSpherix, NY which allows culture and manipulations of the cells under a constant hypoxic condition.  This system has allowed a demonstration of cooperative actions of HIF-1 and NICD1 in Nos2 gene activation under hypoxia (2).
6. Mesothelial cell (MC) isolation and culture:  The first isolation and culture of mouse liver MC, have recently been accomplished by Asahina, the ILCC Associate Director (3). He has identified Glycoprotein M6A (GPM6A) as a specific marker for MC. Using antibodies against GPM6A, MC can be isolated from normal or injured mouse livers by MACS. In culture, MC form epithelial colonies. MC begin to lose the epithelial phenotype and differentiate into fibroblastic cells from Day 4 (click here to see figure). Quantitative RT-PCR shows expression of Gpm6a and Msln (Mesothelin) as markers for MCs (click here to see figure). In parallel to the morphologic changes of “mesothelial-mesenchyme transition”, cultured MC increase their expression of Acta2 mRNA, a marker for myofibroblasts (click here to see figure) .

If you are interested in the ILCC specialized services, please contact Stephanie Pan (stephaqp@usc.edu or 323-442-3850) in advance for the availability of the services, scheduling, and assessment of chargeback rate.

In addition to the routine and customized cell isolation services, ILCC has recently established a Cell Bank that ultimately utilizes the ILCC’s resources and provides NIAAA funded investigators with variety of low-cost cells. The Cell Bank is based on collected and stored fresh or cultured primary cells isolated by the core from the liver of normal or diseased rodent models of liver injury.

ILCC Cell Bank also has limited RNA extracts from the fresh isolated or cultured primary cells from normal or diseased mouse/rat models of liver injury. In general, we will provide a small quantity of the RNA (200-500 ng) free of charge for the purpose of a pilot or feasibility study. If larger quantity is needed, we recommend the investigators to request for the frozen cells through the Cell Bank.

The cells currently available through the Cell Bank include mouse or rat HC, HM, HSC, and LSEC as snap-frozen cell pellets stored at -80oC. Disease models include IG alcohol and control mice, CCl4 or BDL mice or rats.  The lists of cells and RNA samples available from the Cell Bank are shown below.

Available Cell and RNA Stocks: Note: all mice are male C57BL/6J and rats are male Wistar unless specified. Days or wk in the parenthesis indicate the duration of respective treatment prior to isolation. Three to 12 vials are stocked for each listed repository.

Abbreviations in the table: Ac, activated; Alc, alcohol; BDL, bile duct ligation; FACS, cells separated by fluorescence-activated cell sorting; Fresh, stored cells without culture; HC, hepatocytes; HM, hepatic macrophages; HSC, hepatic stellate cells; iG, intragastric infusion model; IM, infiltrating macrophages; KC, Kupffer cells/hepatic macrophages; MACS, cells separated by magnetic-activated cell sorting; PH, partial hepatectomy; Q, quiescent; SEC, sinusoidal endothelial cells.

Rat Cells	Mouse Cells
Click to download	Click to download

REQUESTS FOR CELL BANK SERVICES

1. The Cell Bank charges a flat rate of $30/vial of cells which contains a minimum of 5×10^6 freshly isolated or 1×10^6 cultured cells. This rate does not include shipping fee.
2. The Cell Bank also has a limited repertoire of RNA samples extracted from freshly isolated or cultured primary cells from normal or disease rodent livers. These samples will be shared in a small quantity (200-500ng) free of charge for the purpose of a pilot or feasibility study.
3. If a larger quantity is required, we recommend that investigators request frozen cells through the Cell Bank or freshly isolated cells from the ILCC.
4. The identical service priority criteria as above will be considered for implementation of the Cell Bank services.
5. For more information or to place an order, please contact the Research Laboratory Specialist, Stephanie Pan (stephaqp@usc.edu).

Khanova E, Wu R, Wang W, Yan R, Chen Y, French SW, Llorente C, Pan SQ, Yang Q, Li Y, Lazaro R, Ansong C, Smith RD, Bataller R, Morgan T, Schnabl B, Tsukamoto H. Pyroptosis by caspase11/4-gasdermin-D pathway in alcoholic hepatitis in mice and patients. Hepatology (Baltimore, Md). 2018;67(5). doi: 10.1002/hep.29645. PubMed PMID: 29108122.

Wu R, Murali R, Kabe Y, French SW, Chiang YM, Liu S, Sher L, Wang CC, Louie S, Tsukamoto H. Baicalein Targets GTPase-Mediated Autophagy to Eliminate Liver Tumor-Initiating Stem Cell-Like Cells Resistant to mTORC1 Inhibition. Hepatology (Baltimore, Md). 2018;68(5). doi: 10.1002/hep.30071. PubMed PMID: 29729190.

Machida K. NANOG-Dependent Metabolic Reprogramming and Symmetric Division in Tumor-Initiating Stem-like Cells. Advances in experimental medicine and biology. 2018;1032. doi: 10.1007/978-3-319-98788-0_8. PubMed PMID: 30362094.

Machida K. Pluripotency Transcription Factors and Metabolic Reprogramming of Mitochondria in Tumor-Initiating Stem-like Cells. Antioxidants & redox signaling. 2018;28(11). doi: 10.1089/ars.2017.7241. PubMed PMID: 29256636.

IC# Aizawa S, Brar G, Tsukamoto H. Cell Death and Liver Disease. Gut and liver. 2020;14(1). doi: 10.5009/gnl18486. PubMed PMID: 30917630.

Baulies A, Montero J, Matías N, Insausti N, Terrones O, Basañez G, Vallejo C, Conde de La Rosa L, Martinez L, Robles D, Morales A, Abian J, Carrascal M, Machida K, Kumar DBU, Tsukamoto H, Kaplowitz N, Garcia-Ruiz C, Fernández-Checa JC. The 2-oxoglutarate carrier promotes liver cancer by sustaining mitochondrial GSH despite cholesterol loading., Redox Biol. 2018 Apr;14:164-177. doi: 10.1016/j.redox.2017.08.022, PMID: 28942194.

Gao B, Ahmad MF, Nagy L, Tsukamoto H. Inflammatory pathways in alcoholic steatohepatitis. Journal of hepatology. 2019;70(2). doi: 10.1016/j.jhep.2018.10.023. PubMed PMID: 30658726.

Choi HY, Siddique HR, Zheng M, Yeh D, Machida T, Winer P, Uthaya Kumar D, Rokan A, Punj V, Sher L, Tahara SM, Liang C, Chen L, Tsukamoto H, Machida K. p53 destabilizing protein skews asymmetric division and enhances NOTCH activation to direct self-renewal of TICs. Nature Commun. 2020;11(1). doi: 10.1038/s41467-020-16616-8. PubMed PMID: 32555153.

Eguchi A, Yan R, Pan SQ, Wu R, Kim J, Chen Y, Ansong C, Smith RD, Tempaku M, Ohno-Machado L, Takei Y, Feldstein AE, Tsukamoto H. Comprehensive characterization of hepatocyte-derived extracellular vesicles identifies direct miRNA-based regulation of hepatic stellate cells and DAMP-based hepatic macrophage IL-1β and IL-17 upregulation in alcoholic hepatitis mice. Journal of molecular medicine (Berlin, Germany). 2020;98(7). doi: 10.1007/s00109-020-01926-7. PubMed PMID: 32556367.

Kikuchi K, Tsukamoto H. Stearoyl-CoA desaturase and tumorigenesis. Chemico-biological interactions. 2020;316. doi: 10.1016/j.cbi.2019.108917. PubMed PMID: 31838050.

Machida K. Cell fate, metabolic reprogramming and lncRNA of tumor-initiating stem-like cells induced by alcohol. Chemico-biological interactions. 2020;323. doi: 10.1016/j.cbi.2020.109055. PubMed PMID: 32171851.

Wu R, Pan S, Chen Y, Nakano Y, Li M, Balog S, Tsukamoto H. Fate and functional roles of Prominin 1 + cells in liver injury and cancer. Scientific reports. 2020;10(1). doi: 10.1038/s41598-020-76458-8. PubMed PMID: 33173221.

Lua I, Balog S, Yanagi A, Tateno C, Asahina K. Loss of lysophosphatidic acid receptor 1 in hepatocytes reduces steatosis via down-regulation of CD36. Prostaglandins Other Lipid Mediat. 2021;156:106577. Epub 2021/06/21. doi: 10.1016/j.prostaglandins.2021.106577. PubMed PMID: 34147666; PMCID: PMC8490298.

Win S, Min RWM, Zhang J, Kanel G, Wanken B, Chen Y, Li M, Wang Y, Suzuki A, Aung FWM, Murray SF, Aghajan M, Than TA, Kaplowitz N. Hepatic Mitochondrial SAB Deletion or Knockdown Alleviates Diet-Induced Metabolic Syndrome, Steatohepatitis, and Hepatic Fibrosis. Hepatology. 2021. Epub 2021/08/01. doi: 10.1002/hep.32083. PubMed PMID: 34331779.

Chen CY, Li Y, Zeng N, He L, Zhang X, Tu T, Tang Q, Alba M, Mir S, Stiles EX, Hong H, Cadenas E, Stolz AA, Li G, Stiles BL. Inhibition of Estrogen-Related Receptor α Blocks Liver Steatosis and Steatohepatitis and Attenuates Triglyceride Biosynthesis. The American journal of pathology. 2021;191(7). doi: 10.1016/j.ajpath.2021.04.007. PubMed PMID: 33894178.

Machida K, Tahara SM. Immunotherapy and Microbiota for Targeting of Liver Tumor-Initiating Stem-like Cells. Cancers. 2022;14(10). doi: 10.3390/cancers14102381. PubMed PMID: 35625986.

Serna R, Ramrakhiani A, Hernandez JC, Chen CL, Nakagawa C, Machida T, Ray RB, Zhan X, Tahara SM, Machida K. c-JUN inhibits mTORC2 and glucose uptake to promote self-renewal and obesity. iScience. 2022;25(6). doi: 10.1016/j.isci.2022.104325. PubMed PMID: 35601917.

Hernandez JC, Yeh DW, Marh J, Choi HY, Kim J, Chopra S, Ding L, Thornton M, Grubbs B, Makowka L, Sher L, Machida K. Activated and nonactivated MSCs increase survival in humanized mice after acute liver injury through alcohol binging. Hepatology communications. 2022;6(7). doi: 10.1002/hep4.1924. PubMed PMID: 35246968.

Osna NA, New-Aaron M, Dagur RS, Thomes P, Simon L, Levitt D, McTernan P, Molina PE, Choi HY, Machida K, Sherman KE, Riva A, Phillips S, Chokshi S, Kharbanda KK, Weinman S, Ganesan M. A review of alcohol-pathogen interactions: New insights into combined disease pathomechanisms. Alcoholism, clinical and experimental research. 2022;46(3). doi: 10.1111/acer.14777. PubMed PMID: 35076108.

Osna NA, Eguchi A, Feldstein AE, Tsukamoto H, Dagur RS, Ganesan M, New-Aaron M, Arumugam MK, Chava S, Ribeiro M, Szabo G, Mueller S, Wang S, Chen C, Weinman SA, Kharbanda KK. Cell-to-Cell Communications in Alcohol-Associated Liver Disease. Frontiers in physiology. 2022;13. doi: 10.3389/fphys.2022.831004. PubMed PMID: 35264978.

Balog S, Fujiwara R, Pan SQ, El-Baradie KB, Choi HY, Sinha S, Yang Q, Asahina K, Chen Y, Li M, Salomon M, Ng S W-K, Tsukamoto H. Emergence of highly profibrotic and proinflammatory Lrat+Fbln2+ hepatic stellate cell subpopulation in alcoholic hepatitis. Hepatology, 2022; Accepted.