title: Establishment and characterization of rat portal myofibroblast cell lines.
abstract: The major sources of scar-forming myofibroblasts during liver fibrosis are activated hepatic stellate cells (HSC) and portal fibroblasts (PF). In contrast to well-characterized HSC, PF remain understudied and poorly defined. This is largely due to the facts that isolation of rodent PF for functional studies is technically challenging and that PF cell lines had not been established. To address this, we have generated two polyclonal portal myofibroblast cell lines, RGF and RGF-N2. RGF and RGF-N2 were established from primary PF isolated from adult rat livers that underwent culture activation and subsequent SV40-mediated immortalization. Specifically, Ntpdase2/Cd39l1-sorted primary PF were used to generate the RGF-N2 cell line. Both cell lines were functionally characterized by RT-PCR, immunofluorescence, immunoblot and bromodeoxyuridine-based proliferation assay. First, immortalized RGF and RGF-N2 cells are positive for phenotypic myofibroblast markers alpha smooth muscle actin, type I collagen alpha-1, tissue inhibitor of metalloproteinases-1, PF-specific markers elastin, type XV collagen alpha-1 and Ntpdase2/Cd39l1, and mesenchymal cell marker ecto-5'-nucleotidase/Cd73, while negative for HSC-specific markers desmin and lecithin retinol acyltransferase. Second, both RGF and RGF-N2 cell lines are readily transfectable using standard methods. Finally, RGF and RGF-N2 cells attenuate the growth of Mz-ChA-1 cholangiocarcinoma cells in co-culture, as previously demonstrated for primary PF. Immortalized rat portal myofibroblast RGF and RGF-N2 cell lines express typical markers of activated PF-derived myofibroblasts, are suitable for DNA transfection, and can effectively inhibit cholangiocyte proliferation. Both RGF and RGF-N2 cell lines represent novel in vitro cellular models for the functional studies of portal (myo)fibroblasts and their contribution to the progression of liver fibrosis.
authors.affiliation: Division of Gastroenterology & Hepatology, University of Arkansas for Medical Sciences, Little Rock, AR, United States of America; Research Service, Central Arkansas VA Healthcare System, Little Rock, AR, United States of America.
authors.forename: Michel
authors.initials: M
authors.lastname: Fausther
authors.affiliation: Division of Gastroenterology & Hepatology, University of Arkansas for Medical Sciences, Little Rock, AR, United States of America; Research Service, Central Arkansas VA Healthcare System, Little Rock, AR, United States of America.
authors.forename: Jessica R
authors.initials: JR
authors.lastname: Goree
authors.affiliation: Division of Gastroenterology & Hepatology, University of Arkansas for Medical Sciences, Little Rock, AR, United States of America; Research Service, Central Arkansas VA Healthcare System, Little Rock, AR, United States of America.
authors.forename: Élise G
authors.initials: ÉG
authors.lastname: Lavoie
authors.affiliation: Division of Gastroenterology & Hepatology, University of Arkansas for Medical Sciences, Little Rock, AR, United States of America.
authors.forename: Alicia L
authors.initials: AL
authors.lastname: Graham
authors.affiliation: Département de Microbiologie-Infectiologie et d'Immunologie, Faculté de Médecine, Université Laval, QC, Canada; Centre de Recherche du CHU de Québec, QC, Canada.
authors.forename: Jean
authors.initials: J
authors.lastname: Sévigny
authors.affiliation: Division of Gastroenterology & Hepatology, University of Arkansas for Medical Sciences, Little Rock, AR, United States of America; Research Service, Central Arkansas VA Healthcare System, Little Rock, AR, United States of America.
authors.forename: Jonathan A
authors.initials: JA
authors.lastname: Dranoff
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D001973:Bromodeoxyuridine; D018929:Cell Culture Techniques / Q000379:methods*; D002460:Cell Line; D005455:Fluorescent Antibody Technique; D015151:Immunoblotting; D008099:Liver / Q000166:cytology*; D058628:Myofibroblasts / Q000166:cytology / Q000502:physiology*; D011168:Portal System / Q000166:cytology*; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D015415:Biomarkers; D001973:Bromodeoxyuridine
references.citation: Lab Invest. 2004 Feb;84(2):153-9
references.pmid: 14688800
references.citation: Curr Pathobiol Rep. 2013 Sep;1(3):225-230
references.pmid: 23997993
references.citation: Comp Hepatol. 2006 Nov 16;5:7
references.pmid: 17109742
references.citation: J Hepatol. 1985;1(6):579-96
references.pmid: 4056357
references.citation: Histochem Cell Biol. 2004 Jul;122(1):41-9
references.pmid: 15221412
references.citation: Biochim Biophys Acta. 2013 Jul;1832(7):948-54
references.pmid: 23470555
references.citation: Nat Rev Gastroenterol Hepatol. 2010 Aug;7(8):425-36
references.pmid: 20585339
references.citation: Front Pharmacol. 2014 Jul 22;5:167
references.pmid: 25100997
references.citation: J Biol Chem. 2005 Jun 17;280(24):22986-92
references.pmid: 15799977
references.citation: Hepatology. 2015 Mar;61(3):1041-55
references.pmid: 25043701
references.citation: J Investig Med. 2002 May;50(3):179-84
references.pmid: 12033282
references.citation: J Biol Chem. 2010 Feb 19;285(8):5428-37
references.pmid: 20022962
references.citation: J Hepatol. 2002 Feb;36(2):200-9
references.pmid: 11830331
references.citation: J Vis Exp. 2012;(64). pii: 3669. doi: 10.3791/3669
references.pmid: 22781701
references.citation: J Cell Biochem. 2004 May 15;92(2):414-23
references.pmid: 15108365
references.citation: Nat Commun. 2013;4:2823
references.pmid: 24264436
references.citation: Gastroenterology. 2008 May;134(6):1655-69
references.pmid: 18471545
references.citation: Hepatology. 2010 Apr;51(4):1438-44
references.pmid: 20209607
references.citation: Acta Histochem Cytochem. 2013 Oct 30;46(5):137-43
references.pmid: 24194627
references.citation: BMC Bioinformatics. 2012;13:134
references.pmid: 22708584
references.citation: Lab Invest. 2002 Mar;82(3):323-33
references.pmid: 11896211
references.citation: Curr Pathobiol Rep. 2014;2(4):209-215
references.pmid: 25401051
references.citation: Gastroenterology. 2014 Jul;147(1):41-7
references.pmid: 24814904
references.citation: Proc Am Thorac Soc. 2012 Jul;9(3):137-47
references.pmid: 22802288
references.citation: J Lipid Res. 2000 Jun;41(6):882-93
references.pmid: 10828080
references.citation: Hepatology. 2007 Oct;46(4):1246-56
references.pmid: 17625791
references.citation: Nat Med. 2013 Dec;19(12):1617-24
references.pmid: 24216753
references.citation: Am J Pathol. 2007 Jun;170(6):1807-16
references.pmid: 17525249
references.citation: Hepatology. 2002 Nov;36(5):1135-44
references.pmid: 12395323
references.citation: Proteomics. 2010 Mar;10(5):1017-28
references.pmid: 20049859
references.citation: Proc Natl Acad Sci U S A. 2014 Aug 12;111(32):E3297-305
references.pmid: 25074909
references.citation: J Gastroenterol Hepatol. 2012 Mar;27 Suppl 2:65-8
references.pmid: 22320919
references.citation: Lab Invest. 1991 Dec;65(6):644-53
references.pmid: 1753710
references.citation: Physiol Rev. 2008 Jan;88(1):125-72
references.pmid: 18195085
references.citation: Gut. 2005 Jan;54(1):142-51
references.pmid: 15591520
references.citation: Curr Pathobiol Rep. 2014 Dec 1;2(4):185-190
references.pmid: 25599013
references.citation: Am J Physiol Gastrointest Liver Physiol. 2013 Mar 15;304(6):G605-14
references.pmid: 23328207
references.citation: Am J Pathol. 2007 Nov;171(5):1529-37
references.pmid: 17884967