Effective repopulation of the liver is usually essential for successful clinical hepatocyte transplantation. .05). The engrafted cells expressed several human hepatic markers such as cytokeratin 8 and 18 and albumin as well as transferrin, UGT1A1, hepatocyte nuclear factor (1, 1, and 4), cytochrome CYP3A1, CCAAT/enhancer binding protein ( and ), and human albumin compared with controls. No inflammatory cells were detected in the livers at 4 weeks after transplantation. The improved repopulation of transplanted cells is usually likely a function of the chemokines to mediate cell homing and retention in the hurt liver and might be an attractive strategy to enhance repopulation of transplanted hepatocytes in vivo. for 10 moments to pellet the cells. All women donating fetal tissue experienced been serologically screened for syphilis, toxoplasmosis, rubella, HIV-1, cytomegalovirus, hepatitis B and C, parovirus, and herpes simplex types 1 and 2. Human fetal GSK1070916 IC50 hepatocytes were isolated by magnetic cell sorting, as explained by us previously [14C18]. The method is usually based on a unfavorable selection of this populace using a depletion cocktail including antibodies to 12 lineage-specific cell-surface antigens: anti-CD2, anti-CD3, anti-CD14, anti-CD16, anti-CD19, anti-CD24, anti-CD36, anti-CD38, anti-CD45RA, anti-CD56, anti-CD66b, anti-glycophorin A. The unfavorable portion was used as fetal hepatocytes. All transfection studies were carried out at the Karolinska Institute using unsorted cells. The cells (8.5 weeks of gestation) were transfected using the pCDM7/EF1/hTERT/PAC plasmid and the Amaxa Nucleofector system (Lonza, Walkersville, MD, http://www.lonza.com) (supplemental online data). We cultured and characterized, in detail, cells from one of the bulk clones obtained (hFL161/hTERT) (supplemental online data). Adult Hepatocytes Adult hepatocytes were freshly isolated from three different organ donors after permission from the relatives. The process for isolation was carried out according to the standard protocol . Isolated hepatocytes were cultured in Williams Medium At the without phenol reddish (Sigma-Aldrich, Stockholm, Sweden, http://www.sigmaaldrich.com), further supplemented with 10% AB serum, 5% (vol/vol) l-glutamine (Invitrogen, Carlsbad, CA, SC35 http://www.invitrogen.com), 5% (vol/vol) PEST, 5% (vol/vol) of nonessential amino acids, 5% (vol/vol) sodium pyruvate, vascular endothelial growth factor (5 ng/ml; Invitrogen), interleukin-6 (2 ng/ml; Invitrogen), hepatocyte growth factor (30 ng/ml; Invitrogen), epidermal growth factor (20 ng/ml; Millipore, Solna, Sweden, http://www.millipore.com), and basic fibroblast growth factor (10 ng/ml; Invitrogen). Freshly isolated cells or cells in passage 2 were used for all analyses. Immunohistochemistry and Immunocytochemistry GSK1070916 IC50 for In Vitro Study New frozen sections (5-m thickness) of adult liver (= 3) and fetal liver (= 3) were used. Immunohistochemistry was performed using the biotin-peroxidase complex method. Briefly, photo slides were incubated with main antibodies CCR1, CCR2, CCR3, CCR4, CCR5, and CCR6 and CXCR1, CXCR2, CXCR3, CXCR4, and CXCR6 (1:200 in phosphate-buffered saline; R&Deb Systems, Oxon, U.K., http://www.rndsystems.com) overnight at 4C. The photo slides were then processed as per standard protocol . Immunofluorescent staining of the GSK1070916 IC50 cultured human fetal liver cells transformed with human telomerase reverse transcriptase (hFL4TERT) was performed, as explained in our earlier study , using antibodies specific for human cytokeratins (CK8, CK18, and CK19), human hepatocyte antigen (1:100; all antibodies from Santa Cruz Biotechnology, Santa Cruz, CA, http://www.scbt.com), human c-Met (1:100; Fitzgerald Industries World, Acton, MA, http://www.fitzgerald-fii.com), -fetoprotein (1:500, Abcam, Cambridge, U.K., http://www.abcam.com), and human albumin (ALB; 1:100; Bethyl Inc., Montgomery, TX, http://www.bethyl.com). Circulation Cytometric Analysis New main human adult hepatocytes and hFL4TERT (passages 6, 24, and 50) were analyzed for chemokine receptor manifestation by circulation cytometry. Unconjugated main antibodies CCR1, CCR2, CCR3, CCR4, CCR5, and CCR6 and CXCR1, CXCR2, CXCR3, CXCR4, and CXCR6 (all from R&Deb Systems) were used. Respective fluorescein isothiocyanate isotype controls were used as unfavorable controls. Further procedures were performed as explained in our earlier study . Details are provided in the supplemental online data (supplemental online Table 2). Cell Migration Assay Recombinant human chemokine ligands (R&Deb Systems) CXCL9, CXCL10, and CXCL11 were used to study chemotaxis of hFL4TERT. A migration assay was assessed with.