Source of human liver specimens
Adult human liver specimens were collected from patients who were undergoing partial hepatectomy or liver transplantation and immediately stored at 4°C in UW solution (the University of Wisconsin solution, Netherlands) to isolate hepatocytes. This study was approved by the Institutional Ethical Review Committee of Renji Hospital, School of Medicine, Shanghai Jiao Tong University, and all participants gave informed consent for the collection of their liver specimens.
Cell isolation and culture
Isolation of human hepatocytes was performed as described previously.(18) Briefly, the liver tissue was perfused through intrahepatic vein with PBS for 15 min at 37 °C and then digested with collagenase IV (Sigma, MO, USA) for 25 min. Next, mechanical destruction and filtration of the liver tissue were performed through a 70-um cell strainer to obtain hepatocytes suspension. Finally, the cell pellet was washed twice using GBSS (Gibco, MA, USA) and centrifuged at 80 g for 10 min. The isolated hepatocytes were cultured in Williams’ Medium E (Gibco, MA, USA) with 10% Fetal bovine serum (FBS, Gibco, MA, USA).
The primary mice hepatocytes were isolated and cultured as previously described.(19) UMSCs were cultured in DMEM/F12 supplemented with 10% FBS. UMSCs between passage 3 (P3) and P5 were used in the following experiments.
Encapsulation of cells in alginate–poly-L-lysine–alginate microcapsules
Hepatocytes and UMSCs were immobilized into alginate–poly-L-lysine–alginate (APA) microcapsules by syringe-extrusion technique as previously reported. Briefly, the hepatocytes and the UMSCs were suspended in 0.9% sodium chloride containing 1.5% alginate (Sigma, MO, USA) at a density of 2-5×106 cells/ml. The cell suspension was forced out through an encapsulator (NISCO, Zurich, Switzerland), and the beads were allowed to gel in a hardening bath buffer containing 100 mmol/L of CaCl2 (Sigma-Aldrich, MO, USA), and 10 mmol/L of MOPS (Sigma-Aldrich, MO, USA). After 10 min of hardening, the beads were washed thrice with PBS, and the microspheres were coated with 0.05% (w/v) poly-L-lysine for 10 min, followed by washing with the buffer containing 0.1% CHES, 1.1% CaCl2 and 0.9% NaCl. Furthermore, the microspheres were exposed to 0.15% sodium alginate for 4 min to form the outer layer of the membrane, and then the droplets were washed with saline. The diameter of the microcapsules was approximately 250-350 µm.
Measurement of albumin and urea production
Microcapsules of human hepatocytes (1 × 105 cells) alone or with UMSCs at a ratio of 10:1 or 5:1 or 2.5:1 were seeded in a 24-well plate. The supernatant was collected from day 2 to day 10. The concentration of albumin and urea was measured using albumin ELISA kit (Bethyl Laboratories, TX, USA) and a urea assay kit (Bioassay System, CA, USA) following the manufacturer’s instructions.
Experimental design and animal groups
Male 8-10 weeks old C57BL/6 mice were purchased from Shanghai SLAC Co. Ltd (Shanghai, China). All animal experiments were approved by the Institutional Animal Care and Use Committee of the Shanghai Jiao Tong University School of Medicine (No. SYKX-2008-0050). For intraperitoneal (i.p) transplantation of microcapsules, the mice were randomly divided into four groups with eight mice per group. The groups were as follows: SHAM (blank control, without administration of LPS and D-gal), CON (control, APA), HEP (hepatocytes, 5×106), UMSC-HEP (UMSCs 2×106 and hepatocytes 5×106 cells), and HNF4α-UMSC-HEP (HNF4α-UMSC 2×106 and hepatocytes5×106 cells). ALF was induced by i.p injection of the combination of LPS (50 μg/kg) and D-gal (800 mg/kg) dissolved in PBS for 24 h after microcapsule transplantation. The HB-EGF-neutralizing antibody (anti-HB-EGF, R&D, MN, USA) and control IgG (AB-108-C, R&D, MN, USA) were reconstituted in sterile PBS and administered to ALF mice (10 μg/injection, i.p)
The enzyme activities of aspartate aminotransferase (AST) and alanine transaminase (ALT) were measured using a standard clinical automatic analyzer (Dimension Xpand; Siemens Dade Behring, Munich, Germany). The levels of TNF-α, CXCL15(a homologue of human IL-8), and LDH were detected by ELISA kits (Nanjing Jiancheng Bioengineering Institute, China) according to the manufacturer’s instructions.
Histopathology, immunohistochemistry and immunofluorescence analyses
Liver tissues were fixed in 10% formalin and embedded in paraffin, and then were cut into 5 μm thick sections. The liver sections were then stained with hematoxylin and eosin (HE) for histopathological examination. The histopathological changes were given scores according to a classic liver injury score standard.19 Immunohistochemistry analyses were performed as previously described.(20) For immunohistochemistry, the liver sections were incubated with primary antibodies against MPO (Abcam, Cambridge, UK) or F4/80 (Abcam). After washing with PBS, the sections were incubated with the biotinylated secondary antibody anti-rabbit IgG (Vector, Peterborough, U.K.). Then the sections were visualized using a Vectastain ABC Kit (Vector) at RT 30 min and developed with 3,3’-diaminobenzidine (DAB) at RT. The imagines were visualized by using an inverted microscope. Manual counts were performed to estimate the number of MPO or F4/80 positive cells in the liver of mice in different groups.
For immunofluorescence analyses, the liver sections were incubated with the following primary antibodies: anti-CD68, anti-CD86 and anti-CD206 (all from Abcam, Cambridge, MA, USA). After washing in PBS, the tissue sections were incubated with corresponding conjugated secondary antibodies. The images were then visualized by using an inverted fluorescence microscope. The quantification results were evaluated in at least six representative visual field for each group in a blinded manner by an experienced pathologist. Image-Pro Plus 6.0 (Media cybernetics, Silver Springs, MD, USA) was used for image analysis.
Protein antibody array was performed to test the secretion factors in the conditioned medium (CM) of HNF4α-UMSCs and UMSCs. The expression levels of 1070 human target proteins in cell supernatant were detected. The procedure was done according to the manual of the manufacturer (Aksomics, Shanghai, China). In brief, the protein concentration of the CM was firstly tested by BCA Protein Assay Kit (Kang Chen Corporation, KC-430, China), followed by adding the CM onto the blocked protein array membranes, and then incubating at room temperature (RT) for 2h. Next, the membranes were washed and incubated with biotin-conjugated antibodies at RT for 2 h, and then reacted with HRP-conjugated streptavidin at RT for 2 h. Finally, the membranes were exposed to X-ray films and developed using a film scanner. The intensity of the signals was quantified by densitometry.
Dual-luciferase reporter assay
The cells were cultured into 96-well plates, and were transfected by Lipofectamine TM 3000 (Invitrogen, CA, USA) with the plasmids of HB-EGF promoters, pWPXL or HNF4α, and internal control PRL-TK reporter plasmid after 24 h. After culturing for 48 h, the luciferase activities of the cells were measured by dual-luciferase reporter assay kit (Promega, WI, USA). The experiment was repeated at least three times.
Flow cytometry analysis and cell sorting
Mice were sacrificed and the livers were minced and tamped by using 70 μm filter, the liver leukocytes were purified with 35% Percoll gradient (GE Healthcare), and then the red blood cells were lysed with RBC lysis buffer (ebioscience, MA, USA). The leukocytes were stained with fixable viability dye (ebioscience) and then incubated with Fc block (BD Biosciences), followed by staining with the antibodies against F4/80 (mouse PE T45-2342), CD11b (mouse FITC M1/70) and CD206 (mouse CD206 Alexa 647 MR5D3) (all from Biolegend, CA, USA). Finally, flow cytometry was performed by Cyto FLEX ﬂow cytometer (Beckman coulter, Fullerton, CA, USA) and analyzed with Flowjo software (Treestar, OR, USA).
Data were presented as means ± SEM (n≥3 experiments), and statistical significance were determined using Student’s t test. * P<0.05, ** P<0.01, ***P<0.005, and **** P<0.001; # P < 0.05 and ## P < 0.01.