Isolation and culturing of human placental mesenchymal stem cells (hPMSCs)
Placentas were obtained from donors at the Hangzhou Red Cross Hospital in China after informed consent had been obtained. The study used a protocol approved by the Research Ethics Committee of the First Affiliated Hospital, School of Medicine, Zhejiang University.
The placental tissue was washed with preheated phosphate buffered saline (PBS, pH 7.2 ± 0.1, GenomSciences, Hangzhou, China), minced, and digested using 0.1% (w/v) collagenase type IV (Invitrogen, USA) at 37°C for 30 min. Recovered cells and digested cell debris were filtered through a 100-μm cell strainer. Mononuclear cells (MNCs) obtained by lymphocyte isolation (GE Healthcare, Ficoll-Pague™ PLUS, Sweden) were cultured in special medium (MesenCult® Human Basal Medium plus MesenCult® Human Supplement, STEMCELL Technologies Inc., Vancouver, Canada) and adjusted to 2 × 106 cells in T25-cm2 tissue culture flasks (Nunc Flasks Nunclon™Δ with Filter Cap, Denmark) maintained in an incubator at 37°C in a humidified atmosphere with 5% (v/v) CO2. Approximately 4 to 6 days later, many colonies had formed. After achieving 60-70% confluence, adherent cells were trypsinized using 0.25% (w/v) trypsin/EDTA (Invitrogen, Carlsbad, CA, USA) and re-plated at a 1:3 dilution. Placenta-derived cells were cultured continuously under the same conditions.
Surface antigen expression
Culture-expanded cells (passages 3 to 6) were washed with PBS-containing 0.5% (w/v) bovine serum albumin (BSA) and their concentration adjusted to 1 × 106 cells/100 μL. Their surface antigen expression was evaluated by incubating with anti-human antibodies against CD90-phycoerythrin (PE), CD13-PE, CD133-PE, CD31- allophycocyanin (APC), CD29-APC, CD73-APC, CD105-APC, CD79B-fluorescein isothiocyanate (FITC), CD34-FITC, CD166-FITC, and CD45-TRI COLOR (PE-Cy5- PC5) (eBioscience, Inc., San Diego, CA, USA). After being labeled with antibodies in the dark at room temperature for 20 min, cells were washed twice with PBS. Two-color flow cytometry was conducted using a Beckman Coulter Cytomics FC 500 MPL (Beckman Coulter, Inc., Los Angeles, CA, USA), and the data were analyzed using MXP software.
Adipogenic and osteogenic differentiation
Adherent cells derived from placenta (passages 2 to 6) were plated at a density of 2 × 103/well on six-well multidishes (Nunclon™Δ Surface, Denmark) and cultured in special conditional adipogenic medium (MesenCult® Human Adipogenic Suppl, STEMCELL Technologies, Inc., Canada) as per the manufacturer's protocol. After 4 weeks, adipogenesis was confirmed by oil red O staining (Sinopharm Chemical Reagent Co., Ltd, Shanghai, China) and lipoprotein lipase (LPL) mRNA detection. Human intraoperative abandoned adipose tissue and hPMSCs cultured in normal growth medium served as controls.
MSCs from placenta (passages 2 to 6) were plated at a density of 2 × 103/well in six- well plates (Nunclon™Δ Surface, Denmark) and expanded in special conditional osteogenic medium (OriCell™ hMSC Osteogenic Differentiation Medium, Cyagen Biosciences, Guangzhou, China) and media was changed twice per week. After 4 weeks, osteogenic differentiation was evaluated by Alizarin Red S staining (Genmed Scientifics Inc., USA, Shanghai, China) and detection of osteopontin (OPN) mRNA. hPMSCs cultured in normal growth medium and immortalized human fetal osteoblastic cells (hFOB 1.19 cell line, Cell Bank of the Chinese Academy of Sciences, Shanghai, China) served as controls.
Cultured placental cells (1 × 104/mL; passages 2 to 6) were plated in T25-cm2 tissue culture flasks (Nunc Flasks Nunclon™Δ with Filter Cap, Denmark) and four-well chamber slides (Nunc Lab-Tek™ Chamber Slide™ System, Denmark) with LG-DMEM (Gibco/Invitrogen, USA) containing 10% (v/v) FBS (Gibco/Invitrogen, USA) for 2 days. Hepatogenic induction was conducted as follows. The medium was changed to IMDM supplemented with 20 ng/mL epidermal growth factor (EGF) and10 ng/mL fibroblast growth factor (FGF) for 2 days. The cells were then treated with IMDM with 20 ng/mL hepatocyte growth factor (HGF), 10 ng/mL FGF, and 0.61 g/L niacin amide and 1% (v/v) insulin-transferrin-selenium (ITS) premix (Sigma-Aldrich, St Louis, MO, USA) for 10 days; the medium was changed every 3 days. Finally, cells were maintained in IMDM containing 20 ng/mL oncostatin M (OSM; St Louis, MO, USA), 1 μmol/L dexamethasone (Dex; Sigma-Aldrich, St Louis, MO, USA), and 1% (v/v) ITS premix for 10 days. For the next few days, cells were cultured with HEPATOZYME-SFM (Gibco/Invitrogen, USA). Cells cultured with low glucose DMEM (LG-DMEM) containing 10% (v/v) FBS served as controls. Albumin (ALB), alpha-fetoprotein (AFP), cytokeratin 18 (CK18), and cytokeratin 19 (CK19) levels were determined by immunohistochemistry and RT-PCR; and urea synthesis, low-density lipoprotein (LDL) uptake, glycogen storage, and cytochrome P450(CYP450) enzymatic activity were assayed to evaluate hepatogenic induction on days 7, 14, 21, 28, and 35 after induction.
Cell samples were fixed in 4% (v/v) paraformaldehyde solution at room temperature for 15 min. After being treated with 0.3% (v/v) hydrogen peroxide (H2O2) and blocked in 5% (m/v) BSA, cells were incubated with diluted primary antibody against anti-human CK18 (2 mg/mL, 1:400; Abcam, UK), human AFP (1:200; Abcam, UK), human CK19 (1:400; Abcam, UK), and human ALB (5 mg/mL, 1:500; Abcam, UK), according to the manufacturer's instructions. The primary antibody was detected using horseradish peroxidase-conjugated anti-rabbit IgG (1 mg/mL, 1:1,000; Abcam, UK) antibody followed by incubation with diaminobenzidine tetrahydrochloride solution (DAB kit, Vector Labs, Burlingame, CA, USA). Cultured human hepatocytes served as positive controls.
Reverse transcriptase-polymerase chain reaction (RT-PCR)
Total RNA of cells was extracted with the Trizol reagent (Invitrogen, USA). The quantity and purity of RNA were estimated by reading absorbance at 260 and 280 nm. cDNA was synthesized using oligo dT primers with the Improm-II™ Reverse Transcription System (Promega, Madison, WI, USA) according to the manufacturer's instructions. The primers for the target products were designed as in Table . Polymerase chain reactions (PCR) were carried out in a PCR thermal cycler (Thermo Hybaid, Waltham, MA, USA). PCR products were electrophoresed on a 1.2% (m/v) agarose gel containing 0.5 μg/mL ethidium bromide for nucleic acid visualization under UV light.
Primers and conditions used for RT-PCR
Glycogen storage staining
Primary human hepatocytes were obtained from the Key Lab of Combined Multi-organ Transplantation, Ministry of Public Health, P. R. China.
Undifferentiated and differentiated hPMSCs and primary human hepatocytes (5 × 105/well) were plated in six-well plates and cultured in either LG-DMEM supplemented with 10% (v/v) FBS or HEPATOZYME-SFM (all from Gibco/Invitrogen, USA). Cells were stained with the periodic acid-Schiff (PAS) staining kit according to the manufacturer's protocol (Genmed Scientifics Inc., Shanghai, China).
Low-density lipoprotein (LDL) uptake assay
Undifferentiated and differentiated hPMSCs and primary human hepatocytes (5 × 105/well) incubated in DMEM containing 10% (v/v) FBS were labeled with DiI-LDL (1,19-dioctadecyl-3,3,39,39-tetramethylllindocarbocyane-low-density lipoprotein, Molecular Probes, Invitrogen, US) at 10 μg/mL for 24 h at 37°C. Cells were harvested, washed with cold PBS, and then stained according to the manufacturer's protocol. Uptake was subsequently evaluated by fluorescence microscopy (Olympus IX81, Japan).
Undifferentiated and differentiated hPMSCs and primary human hepatocytes (5 × 105/well) were cultured for 24 h in HepatoZYME-SFM (serum-free, no phenol red, Gibco®, Invitrogen™, USA) supplemented with 0.9 mmol/L ammonium chloride (NH4Cl, Bio Basic Inc., NY, USA). Supernatants were collected, centrifuged, and urea levels were measured in 96-well plates (Nunclon™Δ Surface, Denmark) at 430 nm by a spectrophotometer (Beckman Coulter® Multimode Detector DTX880, Beckman Coulter, Inc.), according to the instructions of the QuantiChrom™ urea assay kit (DIUR-500, BioAssay Systems, CA, USA).
Determination of cytochrome P450 activity
Undifferentiated and differentiated hPMSCs and primary human hepatocytes (5 × 105/well) were plated in six-well plates and cultured in either LG-DMEM supplemented with 10% (v/v) FBS or HEPATOZYME-SFM (all from Gibco/Invitrogen, USA), both with 10 μM rifampicin for 48 h. The media were then changed to IMDM containing a luminogenic CYP substrate (luciferin-PFBE, 50 μM, 1:40 dilution) for 24 h. Intracellular CYP enzyme activities were assessed by assaying luciferin production, according to the manufacturer's instructions (P450-Glo™ CYP3A4 assay with leciferin-PFBE, Promega, USA). Luciferase activity was expressed as relative luminescence units (RLU).
hPMSCs from the placenta (passages 3 to 6) were seeded in T175-cm2 tissue culture flasks (Nunc Flasks Nunclon™Δ with Filter Cap, Denmark). After achieving 70-80% confluence, cells were irradiated with 30 Gy at room temperature using six MV X-rays (500 Mu/min, total of 2890 Mu) from a Varian 23EX Linear Accelerator.
The lethal effect of irradiation on hPMSCs was determined using an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Each group of cells was seeded at a density of 3000 cells per well in 96-well plates (Nunclon™Δ Surface, Denmark). After irradiation for 0, 24, 48, 72, 96, 120, or 144 h, 20 μL of MTT reagent (5 mg/mL, Sigma-Aldrich) was added to each well, and cells were incubated for 4 h at 37°C and 5% (v/v) CO2. The formazan complex was dissolved in 150 μL dimethylsulphoxide (DMSO, Sigma-Aldrich), and absorbance at 490 nm was measured using a spectrophotometer (Beckman Coulter® Multimode Detector DTX880, Beckman Coulter, Inc.). Experiments were performed in triplicate and repeated at least three times. In cell growth curves, the optical density (OD) values were plotted as a function of elapsed time.
Cell-cycle and apoptosis analysis
The effect of irradiation on cell-cycle progression and apoptosis of hPMSCs was investigated by flow cytometry (Beckman Coulter, Inc.). Cell-cycle and apoptosis analyses were carried out at 0 and 6 h and at 3 and 7 days after irradiation treatment. For cell-cycle studies, cells were fixed with pre-cooled absolute ethanol (-20°C) and finally stained using a cell-cycle staining kit (MultiSciences, Hangzhou, China), according to the manufacturer's protocol. The distribution of cell-cycle phases with different DNA contents was determined by flow cytometry and analyzed using multicycle ANOVA analysis software. Cell-apoptosis analysis was carried out by flow cytometry using the Annexin V/PI apoptosis kit (MultiSciences, Hangzhou, China). The percentages of apoptotic cells were analyzed using MXP cytometer software. Experiments were repeated three times.
D-galactosamine-induced acute liver-failure (ALF) model
Male Chinese experimental miniature pigs weighing 10-12 kg were obtained from Beijing Agriculture University (Beijing, China). Animal procedures were approved by the Animal Ethics Committee of Zhejiang University. All pigs were housed in an air-conditioned room with a 12 h dark/light cycle and received humane care for the duration of the study. For catheterization through the external jugular vein, ketalar (0.2 mL/kg) (Fujian Gutian Pharmaceutical Co., Ltd., Fujian, China) was injected intramuscularly to induce sedation. An auricular vein was secured and 2.5 mg/kg/h diprivan (AstraZeneca, Caponago, Italy) administered to achieve continuous anaesthesia. Catheterization was performed as described previously [32
]. Six hours after the operation, acute liver injury in the pigs was induced by administration of D-galactosamine (GalN, Hanhong Chemical Co., Ltd, Shanghai, China) in 5% (w/v) glucose (pH 6.8) at a dose of 1.5 g/kg body weight via a venous catheter. Ultimately, all pigs without cell transplantation died due to GalN-induced hepatic failure, making sacrifice unnecessary. GalN-induced liver injury was identified histologically by a trained pathologist. Animals were considered to be dead when their heartbeat and breathing ceased and blood pressure could not be measured.
Experimental groups and cell transplantation
Twenty-four ALF pigs were randomly divided into four groups (n = 6 each): Group I, ALF pigs without cell transplantation; Group II, hPMSCs transplantation via the jugular venous catheter; Group III, X-ray-treated hPMSCs transplantation via the portal vein; and Group IV, hPMSCs transplantation via the portal vein.
Eighteen hours after GalN administration, hPMSCs (passages 3 to 6; 1.0 × 108 in 5 mL saline) were directly transplanted into the pigs via the jugular venous catheter or the portal vein. For intra-portal cell transplantation, an 18 G PTC needle (Hakko Co., Chikuma-Shi, Japan) with a guide wire was inserted into the left branch of the portal vein inside the liver. The procedure was performed under general anesthesia and guided by color ultrasound (Sequoia 512, Siemens, Germany). By injecting contrast medium SonoVue (Bracco, Milan, Italy), the transplanted cells were seen to be distributed throughout the liver. When the guide wire was withdrawn, blood flow confirmed that the portal vein had been accessed. Then, the hPMSCs preparation was directly and slowly infused into the liver via the portal vein over about 5 min. For jugular vein cell transplantation, the same number and volume of hPMSCs were infused into the jugular vein via a venous catheter. All transplanted pigs were immunosuppressed by daily administration of dexamethasone (Hubei Tianyao Pharmaceutical Co., Ltd, Xiangfan, Hubei) (10 mg/kg/d) during the first week after the transplantation. Blood samples were collected for biochemical analysis at the following time points: pre-and post-GalN administration (18 h), 1 to 7 days after cell infusion, then every 2 weeks from the second week until death. Liver samples from surviving animals were harvested every 2 weeks post-transplantation using an automatic biopsy needle, and the livers of dead animals were directly removed by dissection. These liver tissues were immediately stored at -80°C for molecular detection or fixed in 10% (v/v) formalin for histological and immunohistochemical analysis.
Blood samples were obtained from each pig and centrifuged for 10 min at 1751 × g (Sorvall® Biofuge Stratos, Thermo, Germany) and serum was collected. Concentrations of liver injury markers, such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total bilirubin(TBIL), and total bile acids (TBA) were measured with an automated biochemical analyzer (Abbott Aeroset, Abbott Laboratories, Chicago, IL, USA). All samples were run in triplicate.
Cytokine level measurement
Supernatants of hPMSCs (passages 3 to 6), hPMSCs co-cultured with 1 × 106 lymphocytes from healthy donors for 24 h and serum from model pigs, were collected, centrifuged, and leukemia inhibitory factor (LIF) levels were analyzed using a human LIF platinum ELISA Kit (eBioscience, Inc., San Diego, CA, USA). Tests were performed according to the manufacturer's protocol and every sample was run in duplicate. LIF concentration (pg/mL) was determined by comparison with the standard curve.
hPMSCs (1 × 106) and their supernatants were co-cultured with PBMC (1 × 106) for 24 h. Interferon-γ (IFN-γ) secretion was then assayed by ELISpot, according to the manufacturer's instructions (human IFN-γ ELISpot PRO kit, MABTECH AB, Sweden). The resulting spots were counted and analyzed using a computer-assisted AID ELISPOT Reader System (Immun Spot®, Cellular Technology Ltd.).
Pathology and immunohistochemistry
Pig livers were harvested at the indicated time points after cell transplantation, fixed in 10% (v/v) formalin, and embedded in paraffin. Sections (5-μm thickness) were deparaffinized, rehydrated, and stained with hematoxylin and eosin (H&E) for routine histology. For immunohistochemical evaluation, the sections were heated in citrate buffer (0.02 mol/L, pH 5.8) for antigen retrieval. Endogenous peroxidase was prevented by immersion in a 0.3% (v/v) hydrogen peroxide (H2O2) in methanol bath for 15 min. After washing, non-specific binding was blocked with 5% (m/v) bovine serum albumin in PBS. Sections were then incubated with diluted primary antibody against anti-human ALB (5 mg/mL, 1:5,000; Abcam, UK), human AFP (1:250; Abcam, UK), and human CK18 (1 mg/mL, 1:200; Abcam, UK), according to the manufacturer's instructions. Detection of primary antibody was performed using horseradish peroxidase-conjugated secondary antibodies (1 mg/mL, 1:1,000; Abcam, UK). Peroxidase activity was revealed by a 3 to 5 min exposure to diaminobenzidine tetrahydrochloride solution (DAB kit, Vector Labs). The sections were then washed, counterstained with hematoxylin for one minute, mounted, and observed under a light microscope (TE2000, Nikon, Japan). Normal human tissue obtained under informed consent served as positive controls.
Detection of human alu sequences by PCR
PCR for human alu sequences was performed to confirm the presence of transplanted hPMSC-derived cells in recipient pig livers. Total DNA of liver samples was extracted with a DNeasy® Blood & Tissue Kit (Qiagen, Hilden, Germany), according to the manufacturer's instructions. The primers used were 5'-CTGGGCGACAGAACGAGATTCTAT-3' and 5'-CTCACTACTTGGTGACAGGTTCA-3'; samples were incubated at 94°C for 2 min and then amplified for 35 cycles of denaturation for 30 s at 94°C, annealing for 30 s at 60°C, and extension for 59 s at 72°C. The PCR products were analyzed by resolution on a 1.2% (w/v) agarose gel and visualized by ethidium bromide staining. Images were captured using a gel documentation system (Syngene GBox-HR Gel Doc System, UK).
Data were presented as the mean ± standard deviation (SD). Differences in serum levels of biochemical parameters were analyzed using a paired-samples t-test. Statistical evaluations of cell viability, cell-cycle distribution, and percentage apoptosis, urea synthesis, CYP-450 enzymatic activity, LIF, and IFN-γ secretion were performed using a t-test. Animal survival was analyzed using the Kaplan-Meier log rank method. Values of P < 0.05 were considered statistically significant. Data were analyzed with SPSS ver. 16.0 statistical software (SPSS, Inc., Chicago, IL, USA).