Human H9 embryonic stem cells (WA09, WiCell, Madison, WI, USA) were cultured on MEFs treated with mitomycin C. Cells were manually dissected and plated onto 0.1% gelatin-coated culture dishes (Costar, Corning Life Sciences, Acton, MA, USA) for amplification. The human ES cell culture medium was composed of standard Dulbecco’s modified Eagle’s medium (DMEM)/F12 supplemented with 20% Knockout Serum Replacement (Gibco/Life Technologies Corp., Saint Aubain 91190, France), 4 ng/ml human recombinant basic fibroblast growth factor (bFGF; R&D Systems Inc., Minneapolis, MN, USA), 0.1 mmol/l β-mercaptoethanol, 1 mmol/l L-glutamine, and 1% non-essential amino acids. Fetal livers were obtained from pregnancies terminated at 11 to 13 weeks of gestation, after obtaining the informed consent of the mothers, as recommended by the French Ethics Committee and the local Ethics Committee of Paris XI University (Paris, France). Human hepatoblasts were then isolated and cultured as previously described [25
]. Primary human adult hepatocytes were isolated from normal liver tissue biopsy specimens obtained during resections (partial hepatectomies) for hepatic or metastatic tumors, after informed consent had been obtained from the patient. MSCs, HeLa cells, human cervical epithelial tumor cells, HuH7cells (a human hepatoma cell line), and COP cells (a cell line generated from SV40 transformation of human pancreatic islets) were used to seed a six-well microplate (CellBind; Corning Life Sciences) containing DMEM (PAA Laboratories, Les Mureaux, France) supplemented with 10% fetal bovine serum (FBS). They were cultured at 37°C for 24 hours, under an atmosphere containing 5% CO2
. Transduction was performed overnight at an MOI of 40 for MSCs and HeLa cells, and at an MOI of 30 for HuH7 cells, in the presence of 8 μg/ml hexadimethrine bromide (Polybrene; Sigma-Aldrich Chimie, Lyon, France). The supernatant was then replaced with fresh culture medium.
A549 cells (a human alveolar epithelial cell line) were used to seed six-well microplates (CellBind; Corning Life Sciences) containing HamF12 (PAA Laboratories) supplemented with 10% FBS, and then cultured at 37°C for 24 hours, under an atmosphere containing 5% CO2. Cells were transduced overnight at an MOI of 40, in the presence of 8 μg/ml hexadimethrine bromide (Polybrene; Sigma-Aldrich). The supernatant was then replaced with fresh culture medium.
MCF7 cells (a breast cancer epithelial cell line) were used to seed six-well microplates (CellBind; Corning Life Sciences) containing MEM (PAA Laboratories) supplemented with 10% FBS + 0.01 mg/ml bovine insulin (Sigma-Aldrich) and were cultured at 37°C for 24 hours, under an atmosphere containing 5% CO2. Cells were transduced overnight at an MOI of 80, in the presence of 8 μg/ml hexadimethrine bromide (Polybrene; Sigma-Aldrich). The supernatant was then replaced with fresh culture medium. At 1 week after transduction, GFP expression was analyzed by flow cytometry or fluorescence microscopy. Phase-contrast images were taken under a microscope (Eclipse; Nikon, Tokyo, Japan).
Human fibroblasts were cultured in DMEM medium supplemented with 10% FBS and antibiotics. HUVECs were cultured in chemically defined EBM2 endothelial basal medium with antibiotics. Cells were transduced overnight at an MOI of 30. At 4 days after transduction, GFP expression was analyzed by flow cytometry. COP cells were cultured in low-glucose DMEM supplemented with 10% FBS and antibiotics.
Differentiation of hESCs into hepatic progenitor cells
At 1 day before the passage of hESCs for differentiation, 30 × 60 mm cell culture dishes (CLS430166-500EA; Corning Life Sciences) were coated with 0.1% gelatin from porcine skin type A (61890-100G; Sigma-Aldrich). After 90 min of incubation at room temperature, the gelatin was removed, and dishes were washed once with phosphate-buffered saline (PBS). A coating medium (DMEM, 10% FBS, 100 × MEM non-essential amino acid solution, 1% L-glutamine) was added to plates, which were then incubated for 24 hours at 37°C, under an atmosphere containing 5% CO2. The following day, hESCs were dissected from MEFs. For this, the hESC culture medium was removed from the cells and replaced with chemically defined medium (CDM) supplemented with bovine serum albumin (BSA; Europabioproducts, Ely, UK; 5 mg/ml final concentration), fibroblast growth factor (FGF)2 (12 ng/ml final concentration) and activin A (10 ng/ml final concentration). About 80 colonies of hESCs on MEFs per 60 mm dish were dissected with a sterile pipette tip. The coating medium was removed from the gelatin-coated dishes, which were washed once with 1 × PBS, then CDM-BSA containing the previously dissected hESC clusters was added to the plates. We used 40 dissected colonies per pre-coated plate. After incubation for 48 hours, the CDM-BSA was removed and replaced with CDM supplemented with polyvinylalcohol (PVA; Sigma-Aldrich; 1 mg/ml final concentration: CDM-PVA), activin A (100 ng/ml final concentration), FGF2 (20 ng/ml final concentration), bone morphogenetic protein (BMP)4 (10 ng/ml final concentration), and LY294002 (10 μmol/l final concentration). The medium was replaced daily. After 3 days, cells were incubated with CDM-PVA supplemented with FGF10 (50 ng/ml) for a further 3 days. Retinoic acid (0.1 μmol/l final concentration) and SB431542 (10 μmol/l final concentration) were then added, together with FGF10, and the cells incubated for an additional 2 days. Finally, cells were incubated for 4 days with CDM-PVA supplemented with hydrocortisone (1 μmol/l final concentration), FGF4 (30 ng/ml final concentration), HGF (50 ng/ml final concentration) and epidermal growth factor (EGF; 50 ng/ml final concentration).
On day 16 of differentiation, attached cells were removed in a cell dissociation buffer (0.1 mg/ml EDTA and 0.5 mg/ml BSA in PBS), and GFP-expressing cells were purified with a cell sorter (FACS DiVa Flow Cytometer; Becton Dickinson, Franklin Lakes, NJ, USA). Purified hepatic progenitors in a plating medium (DMEM/HAM F-12, 10% heat-inactivated FBS, 1 g/l human BSA fraction V, 1% L-glutamine) were plated onto a type I collagen-coated plate and incubated for 4 hours. Cells were then incubated overnight with hepatic progenitor medium (HPM: DMEM/HAM F-12/Williams Medium E 1:1, 0.24% linoleic acid-albumin 2:1, 5.10-8 mol/l triiodo-L-thyronine, 0.2 IU insulin, 10-6 mol/l hydrocortisone, 6 × 10-4 mol/l vitamin C, 6 × 10-4 mol/l human apo-transferrin) supplemented with HGF (50 ng/ml). For the next 2 days, cells were incubated with HPM supplemented with HGF (50 ng/ml) and EGF (20 ng/ml). Cells were then incubated with hepatocyte culture medium (Lonza Group Ltd, Basel, Switzerland) supplemented with the associated kit (HCM Bullet Kit; Lonza) and Oncostatin M (10 ng/ml). Phase-contrast images were taken under a microscope (Eclipse; Nikon).
The APOA-II promoter was previously cloned in lentivectors by our laboratory [12
]. The EF1α-GFP and APOA-II-GFP cassettes were inserted into third-generation self-inactivating (SIN) lentivectors containing a WPRE sequence and a mutated GAG sequence. These vectors were designed and produced by Vectalys SAS (Toulouse, France).
Viral vectors were produced in a human embryonic kidney (HEK)293T cell line. The HEK293T cells were used to seed a 10-layer cell culture chamber (6320 cm2; CellSTACK; Corning Life Sciences) and were transfected 2 days later, in fresh DMEM without fetal calf serum (FCS) supplemented with 1% penicillin/streptomycin and 1% ultraglutamine (PAA Laboratories). Cells were simultaneously transfected with three plasmids: pVSVG, pGagPol, and pLV-APOA-II-GFP. The supernatant was discarded 24 hours after transfection, and replaced with fresh non-supplemented DMEM. The harvested vectors were clarified by centrifugation for 5 minutes at 3000 g, followed by microfiltration through a sterile filter unit with 0.45 μm pores (Stericup; Millipore Corp., Billerica, MA, USA). The crude vector preparation was concentrated and purified by tangential flow ultrafiltration, and the supernatant was then diafiltered against DMEM. Once the diafiltration was complete, the retentate was recovered, and further concentrated by ultrafiltration.
Quantification of functional particle by FACS
HCT116 cells were used to seed 96-well plates at a density of 12,500 cells per well, in 250 μl of DMEM supplemented with 10% FCS, 1% penicillin/streptomycin, and 1% ultraglutamine (complete medium). Five serial dilutions with complete medium were performed 24 hours later for each vector sample and an rLV-EF1-GFP internal standard. The cells were transduced with these serial dilutions in the presence of 8 μg/ml hexadimethrine bromide (Polybrene; Sigma-Aldrich). For each sample series, one well of non-transduced cells was included as a control. At 4 days after transduction, the cells were released by trypsin treatment and harvested by centrifugation, then each cell pellet was resuspended in 250 μl of PBS. The titer was calculated by determining the number of transducing units (TU)/ml by FACS.
Quantification of physical particles by p24 ELISA
The p24 core antigen was detected directly in the viral supernatant with a HIV-1 p24 ELISA kit (Perkin Elmer, Waltham, MA, USA) in accordance with the manufacturer’s instructions. The absorbance of each microplate well was determined with a microplate reader, and calibrated against tan HIV-1 p24 antigen standard curve. The viral titer, expressed in physical particles per ml, was calculated from the amount of p24, assuming that 1 pg of p24 corresponds to 104 physical particles.
Transduction of hESCs by lentivectors
Before transduction, hESCs were manually dissociated and incubated, in clumps, with viral particles for 2 hours at 37°C in low-attachment 24-well plates (Corning Life Sciences), with gentle rocking. They were then added to MEFs in hESC medium. The undifferentiated transduced cell population was expanded and differentiated in CDM [12
] devoid of serum and supplemented with insulin, transferrin, and defined lipids, to which was added BSA (5 mg/ml fraction V; Europabioproducts) for expansion or PVA (1 mg/ml, mean molecular weight 30,000 to 70;000; P8136-250G, Sigma) as a substitute for BSA.
Transduction of human ESC-derived hepatic progenitor cells by IDLV
On day 13 of differentiation, cells were washed once with PBS, and fresh CDM-PVA supplemented with HGF (50 ng/ml final concentration), EGF (50 ng/ml final concentration), FGF4 (30 ng/ml final concentration), and hydrocortisone (10-6 mol/l final concentration) were added. The IDLV was used at an MOI of 30 and was incubated with cells for 24 hours. The cells were cultured for a further 2 further days, with the medium changed daily. The HIV integrase inhibitor raltegravir was added to the culture medium on the day of transduction, at a concentration of 1 μmol/l, and was maintained in the medium for 24 hours.
Cell preparation for sorting and plating
Differentiated cells were washed with PBS and incubated for 5 min at 37°C with 2 ml per 60 mm dish of cell dissociation buffer (0.01 mg/ml EDTA, 0.05 mg/ml BSA in PBS without Ca2+/Mg2+, pH 7.5). Dissociated cells were suspended in 5 ml of plating medium (DMEM/F12; 20% human serum, PAA Laboratories), and separated by centrifugation for 5 minutes at 400 g. The cell pellet was finally suspended in plating medium (DMEM/F12, 20% human serum 1 mmol/l L-glutamine, 100 μl/ml antibiotics) supplemented with HGF (50 ng/ml final concentration), at a density of 3 × 106 cells/ml for FACS. GFP-positive cells were plated onto a 24-well type I collagen-coated plate (BD Biosciences) at a density of 2 × 105 cells per well. At 4 hours after plating, the medium was replaced with CDM-PVA supplemented with HGF (50 ng/ml final concentration), EGF (50 ng/ml final concentration), and FGF4 (30 ng/ml final concentration), and cells were cultured for a further 2 days before analysis. Phase-contrast image were taken under a microscope (Eclipse; Nikon).
Southern blot hybridization was carried out using digoxigenin (DIG System; Roche Applied Science, Basel, Switzerland) in accordance with the manufacturer’s protocol. Briefly, 5 μg of genomic DNA from each sample was digested with either Eco
RI (New England Biolabs, Beverly, MA, USA) for the detection of 1- and 2-LTR circular DNA (4,396 bp and 4,627 bp bands, respectively) or with Eco
RI and Bam
HI (New England Biolabs) for the detection of total lentiviral DNA as a 1,403 bp band. The digested DNA was subjected to electrophoresis in a 1% agarose gel. Gels were blotted onto Hybond-N + membrane (Millipore) overnight in 20 × SSC buffer (Sigma-Aldrich). As a probe, we used 1.3 kb PCR (GFP-WPRE) fragments (see Additional file 1
: Figure S1B) labeled with DIG-11-dUTP (PCR DIG Probe Synthesis Kit; Roche). The probe was hybridized with the membrane overnight at 42°C (DIG Easy Hyb solution; Roche), and the probe detected ()DIG Detection System; Roche). The DIG-labeled probe was detected with an anti-digoxigenin-AP Fab fragment (Roche) and visualized with achemiluminescent substrate (CSPD; Roche).
Evaluation of copy number
The copy number of the lentiviral vector was determined by qPCR analysis. The qPCR experiments were performed with 150 ng of total DNA, SYBR GreenER (Invitrogen Corp., Carlsbad, CA, USA), specific primers binding to the WPRE sequence, and albumin (housekeeping gene) in a final volume of 20 μl (Step One Real-Time PCR System; Applied Biosystems, Foster City, CA, USA). Copy number was calculated by referring the Ct values for each sample to a standard plasmid curve. All qPCRs were performed in duplicate.
To determine the limits of detection of residual lentivector integration, serial dilutions were performed using genomic DNA from a clonal cell line containing only one copy of ILV. The line was established by transduction of HCT116 cells with a GFP-expressing ILV, followed by clonal selection, and the copy number was quantified by Southern blotting. Serial dilutions of genomic DNA bearing one copy per cell were performed by two-fold dilutions in genomic DNA from control non-transduced HCT116 cells. A range of dilutions from 1:2 to 1:10000 were tested. All qPCRs were performed in duplicate.
Total RNA was extracted (RNeasy Mini Kit (Qiagen Inc., Valencia, CA, USA) from hESCs, differentiated hepatic progenitors, and human fetal and adult hepatocytes, following the manufacturer’s protocol. For each sample, 0.6 μg of total RNA was reverse-transcribed with reverse transcriptase (Superscript II; Invitrogen), and amplification by PCR was performed (PCR Nucleotide Mix Kit; Promega, Charbonnières, France). The primers used and the sizes of the amplicons obtained are described in Table .
Primer sequences used for reverse transcription-PCR in this study
PCR and real-time qPCR
Real-time PCR mixtures were prepared (SensiMiX Kit; Bioline, Paris, France), in accordance with the manufacturer’s instructions. Primers used for real-time PCR analyses are shown in Table . The DNA was then denatured at 95°C for 10 minutes, and subjected to 40 cycles of 95°C for 30 seconds, 60°C for 30 seconds, and 72°C for 30 seconds, followed by a final extension at 72°C for 10 minutes. Real-time PCR was performed in a real-time PCR system (model 7300 Applied Biosystems), in triplicate, with normalization to hypoxanthine-guanine phosphoribosyltransferase (hPRT) levels in the same run. The real-time qPCR results are presented as the means of three independent experiments; error bars indicate the SEM.
Quantitative PCR primer sequences used in this study
FACS analysis of sorted cells
After transduction with the IDLV ApoA-II-GFP lentivector, the differentiating hepatocytes were analyzed for GFP expression 14 days after progenitor sorting, using a cytometer (n Accuri C6; Becton Dickinson) and associated software (CFlowPlus; Becton Dickinson).
Transduction of purified progenitors
Purified progenitors were transduced 5 days after sorting, with the Cyp3A4-GFP lentivector (kindly provided by Dr Anne Corlu, INSERM U 991), and fluorescence was assessed 12 days later. Rifampicin (10 μmol/l) or DMSO was added to the plates at day 30 for 48 hours, and cells were analyzed 1 day later (day 33).
Cells were fixed by incubation with 4% paraformaldehyde for 10 minutes at room temperature, then washed with 50 mmol/l NH4Cl in PBS for 10 minutes, and permeabilized by incubation with 0.1% Triton X-100 for 4 minutes. Cells were blocked by incubation with 3% serum in PBS for 1 hour. Primary antibodies were diluted in PBS supplemented with 4% BSA (see Table for the corresponding dilution) and incubated with cells for 2 hours at room temperature. Cells were then washed with PBS and incubated with secondary antibodies diluted in PBS supplemented with 4% BSA (see Table for corresponding dilution) for 1 hour at room temperature. Cell nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI), and cells were mounted in (Vectashield Mounting Medium; Vector Laboratories/AbCys, Paris, France). Fluorescence micrographs were obtained with AxioVision Rel. 4.8 microscopy software.
Antibodies used in this study
ICG uptake and release
The ICG uptake test was performed on cells 17 days after sorting, by incubating the cells with 1 mg/ml ICG for 60 min at 37°C. Cells were then washed in medium, and ICG release was evaluated 16 hours later.
Albumin concentrations in cell culture medium were measured with a kit specific for human albumin detection, in accordance with the manufacturer’s instructions (Dade Behring SAS, France) in the Department of Biochemistry, Bicêtre Hospital.
Glycogen storage was assayed by the periodic acid-Schiff technique according to McManus [33