Integrative Liver Cell Core

THE INTEGRATIVE LIVER CELL CORE (ILCC), formerly known as the Non-Parenchymal Liver Cell Core…

• Strives to serve the scientific community of alcoholic liver disease (ALD) and cirrhosis via specialized services involving isolation of different liver cell types from normal rodents and rodent models of ALD and liver fibrosis.
• Provides primary or stored hepatocytes (HC), hepatic macrophages/Kupffer cells (HM), hepatic stellate cells (HSC), liver sinusoidal endothelial cells (LSEC), liver mesothelial cells (MC), CD133+ liver tumor-initiating stem cell-like cells (TICs), and CD133+ liver progenitor cells, each isolated from mice or rats.
• Supports specialized analysis such as cell lineage and fate tracing using Rosa-reporter mice; FACS-based isolation of quiescent vs. activated HSCs from Col1a1-GFP mice; infiltrating vs. resident HM and tracking on blood monocytes.
• Offers Cell Bank, a collection of small aliquots of isolated cells or RNA from normal and diseased rodent livers for nominal fees – ideal for pilot analysis.

A. Primary mouse HSCs and their vitamin A autofluorescence.
B. Primary rat HMs.
C. Primary mouse LSECs and their uptake of Acetylated-LDL.
D. Primary mouse MCs.

Unique services of the Integrative Liver Cell Core (ILCC)

Isolation and analysis of 6 liver cell types; hepatocytes (HC), CD133+ ductular reaction progenitors (DRP), hepatic macrophages (HM), sinusoidal endothelial cells (SEC), hepatic stellate cells (HSC), and liver mesothelial cells (MC) from 12 different mouse models of alcoholic liver disease (ALD), alcohol-promoted liver tumor, and liver fibrosis. These services are integrated with cell lineage and fate tracing techniques using Rosa26-reporter mice or loss of gain of function approaches using conditional knock-out or knock-in genetic mice to support research on altered cell fate regulation of these different cell types and the specific role of a gene of interest in a particular cell type in evolution of the diseases.

During 2015-2020, the ILCC has served 41 investigators from 25 institutions by performing 1,151 isolation preparations and providing 283 cell repository samples from the Cell Bank. These efforts culminated to supporting publication of 34 articles, acquisition or continuation of 20 federal grants of which 11 are from NIAAA. The ILCC has served as a national resource by providing the services to 22 out-of-region investigators across the nation of which 14 were early career investigators.

Cell Bank Service

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.

ILCC laboratory staff

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.

ILCC brochure (Website version). Contact Felicia Owusu, MPA to receive hard copies.

Requests For Routine Or Common Services

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 Management And Operations

Director: Hidekazu Tsukamoto, D.V.M., Ph.D. (htsukamo@med.usc.edu)

ILCC Supervisor: Stephanie Pan (stephaqp@usc.edu)

Core Management (Chargebacks): Aileen Luminario (Aileen.Luminario@med.usc.edu)

Publications Supported By ILCC:

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.