hescs (H1 and H9) and ipscs (3U1) were maintained and directed into definitive endoderm

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1 Supplementary information, Data S1 Materials and Methods Culture and differentiation of hpscs hescs (H1 and H9) and ipscs (3U1) were maintained and directed into definitive endoderm cells as described previously. Further differentiation was carried out as indicated in Figure 1A in RPMI 1640 (Gibco) supplemented with Glutamax, 0.5 mg/ml albumin fraction V (Sigma-Aldrich) and 1% insulin-transferrin-selenium (ITS) (Sigma-Aldrich) on days 4 and 5, or DMEM/F12 (Gibco) supplemented with Glutamax, 0.1 mm b-mercaptoethanol, 1% nonessential amino-acids and 1% B27 supplement (Gibco) from day 6 to day 15, or hepatocyte culture medium supplemented with SingleQuots (Lonza) from days 16 to 20. The day 5 ventral foregut cells were dissociated to single cells by Accutase (Millipore) and replated at a density of /ml ( /cm 2 ) on 50-fold-diluted Matrigel Matrix Growth Factor Reduced (Becton Dickinson) coated plates. The cell viability after replating is usually more than 90%. Recombinant human bfgf (4 ng/ml), activin A (100 ng/ml), FGF7 (20 ng/ml), BMP2 (20 ng/ml), and HGF (20 ng/ml) were from Peprotech. SB (10 mm) was from Tocris Bioscience. Recombinant human BMP4 (50 ng/ml) and oncostatin M (10 ng/ml) were from R&D Systems. Dexamethasone (Dex, 0.1 μm) was from Sigma-Aldrich. Albumin and alpha-1-antitrypsin secretion Human albumin or alpha-1-antitrypsin levels in culture supernatants were measured using the Human Albumin ELISA Quantitation kit (Bethyl Laboratory) or an alpha-1-antitrypsin clearance EIA kit (Alpco Diagnostics). The content of total protein was measured using the Pierce BCA protein Assay kit (Thermo Scientific). Albumin and alpha-1-antitrypsin secretion

2 was normalized to total cell protein. Fresh human hepatocytes isolated according to standard two-step EDTA/collagenase perfusion protocol and cryopreserved 25-week fetal hepatocytes were used as control and cultured in hepatocyte culture medium supplemented with SingleQuots (Lonza) for 12 hours prior to their use in the studies. Basal metabolic activity, induction and inhibition assays To determine the basal activity (without inducers) of metabolic enzymes, cultures were incubated with substrates at 37ºC as follows: phenacetin at 100 µm, diclofenac at 100 µm, S-mephenytoin at 100 µm, chlorzoxazone at 300 µm, testosterone at 200 µm, 7-hydroxycoumarin at 50 µm and azidothymidine at 50 µm. All substrates were from Sigma-Aldrich except S-mephenytoin (Becton Dickinson). All substrates were incubated for 6 h except phenacetin and chlorzoxazone for 24 h. The reactions were stopped by collection of the incubation medium and mixed with 3-fold volumes of methanol for further measurements. For induction assays, cultures were treated for 72 h with the 50 µm omeprazole or vehicle control (0.1% DMSO) with daily renewal of the culture medium plus freshly added omeprazole or vehicle control. CYP1A2 activity was measured at the end of the 72 h treatment period. For inhibition assays, cultures were incubated with substrates in the presence of specific inhibitors (furafylline for CYP1A2 and ketoconazole for CYP3A4). At least nine different concentrations of each inhibitor were tested. The concentrations of the metabolites in the supernatants were measured by Pharmaron Co. Ltd. using validated traditional LC/MS/MS methods. The metabolites were used to make standard curves for the metabolite analyses. Isotope-labeled reference metabolites were used as internal standards.

3 The production of metabolites was normalized to total cell protein. Toxicity assays Cultures were treated for 24 h with various concentrations of troglitazone, rosiglitazone or vehicle (DMSO). Cell viability was subsequently measured by the standard MTT assay. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was purchased from Sigma-Aldrich. Characterization of Canalicular Transport The live cultures were washed three times with PBS, then incubated with 2 μg/ml CDF [5-(and-6)-carboxy-2',7'-dichlorofluorescein diacetate, Invitrogen] or 5 μm CLF (cholyl-lysyl-fluorescein, Becton Dickinson) for 10 min, and washed three times again prior to examination with fluorescence microscopy. Immunofluorescence staining Cultures and tissue sections were fixed for 15 min at room temperature in 4% paraformaldehyde in PBS, washed several times in PBS, and blocked for 60 min in PBS containing 5% normal donkey serum (Jackson ImmunoResearch Laboratories) and 0.1% Triton X-100 (Sigma-Aldrich). Primary antibodies were diluted in PBS containing 5% normal donkey serum and 0.1% Triton X-100 and incubated at 4 C overnight. Secondary antibodies were diluted in PBS containing 1% bovine serum albumin and incubated for 1 h at room temperature. DAPI (1 μg/ml; Roche) was used to stain the cell nuclei. Primary antibodies

4 were purchased from the following: mouse anti-afp, rabbit anti-aat, mouse anti-krt18 (Invitrogen); rabbit anti-hnf4a, rabbit anti-ttf1, rabbit anti-cebpb, goat anti-hnf1a, goat anti-gata4, goat anti-hnf1b, mouse anti-asgpr, mouse anti-cypor (Santa Cruz Biotechnology); mouse anti-hnf4a, rabbit anti-prox1, mouse anti-mrp2 (Abcam); goat anti-prox1, goat anti-tbx3, goat anti-foxa1, goat anti-pdx1, sheep anti-hnf6 (R&D systems); rabbit anti-cyp3a4,rabbit anti-cyp1a2 (AbDSerotech);mouse anti-alb (Cedarlane Labs); rabbit anti-cepba (Cell Signaling Technology); mouse anti-sox17 (Origene). Isotype-matched antibodies served as negative controls (Supporting information, Figure S10). Real-time quantitative PCR Total RNA was isolated from cells using an RNeasy micro or mini kit (Qiagen). Genomic DNA contamination was eliminated by an on-column DNase treatment step using an RNase-Free DNase Set (Qiagen). Total RNA (1 μg) was used for reverse transcription with a SuperScript III first-strand synthesis system (Invitrogen). Real-time RT-PCR analysis was performed on an ABI Prism 7300 Sequence Detection System using the SYBR Green PCR Master Mix (Applied Biosystems). The relative expression of each gene was normalized against GAPDH. Primer sequences were as following:gapdh, 5 -TGCACCACCAACTGCTTAGC-3 (forward) and 5 -GGCATGGACTGTGGTCATGAG-3 (reverse); OCT4, 5 -TTCAGCCAAACGACCATCTG-3 (forward) and 5 -TCAGCTTCCTCCACCCACTT-3 (reverse);sox2, 5 -GCACATGAAGGAGCACCCGGATTA-3 (forward) and

5 5 -CGGGCAGCGTGTACTTATCCTTCTT-3 (reverse); SOX17, 5 -GCATGACTCCGGTGTGAATCT-3 (forward) and 5 -TCACACGTCAGGATAGTTGCAGT-3 (reverse); FOXA2, 5 -CTGAGCGAGATCTACCAGTGGA-3 (forward) and 5 -CAGTCGTTGAAGGAGAGCGAGT-3 (reverse); GATA4, 5 -CGACACCCCAATCTCGATATG-3 (forward) and 5 -GTTGCACAGATAGTGACCCGT-3 (reverse); HNF1B, 5 -GCACCTCTCCCAGCATCTCA-3 (forward) and 5 -GTCGGAGGATCTCTCGTTGC-3 (reverse); HNF4A, 5 -ACTACATCAACGACCGCCAGT-3 (forward) and 5 -ATCTGCTCGATCATCTGCCAG-3 (reverse); TBX3, 5 -CCCGGTTCCACATTGTAAGAG-3 (forward) and 5 -GTATGCAGTCACAGCGATGAAT-3 (reverse); PROX1, 5 -CAATTTCCACACCGCCAAC-3 (forward) and 5 -TCAGTGGAACTGGCCATCTG-3 (reverse); HNF6, 5 -TGTGGAAGTGGCTGCAGGA-3 (forward) and 5 -TGTGAAGACCAACCTGGGCT-3 (reverse); AFP, 5 -CCCGAACTTTCCAAGCCATA-3 (forward) and 5 -TACATGGGCCACATCCAGG-3 (reverse); CEBPA, 5 -AGAAGTCGGTGGACAAGAACAGCA-3 (forward) and 5 -ATTGTCACTGGTCAGCTCCAGCA-3 (reverse); HNF1A, 5 -GGTCCTACGTTCACCAACACA-3 (forward) and 5 -CTCTGGGTCACATGGCTCT-3 (reverse); ALB, 5 -CTAGGAAAAGTGGGCAGCAAATG-3 (forward) and 5 -GCGTTTTCTCATGCAACACACAT-3 (reverse); AAT, 5 -GAGGCTCAGATCCATGAAGG-3 (forward) and 5 -GGTGTCCCCGAAGTTGACAG-3

6 (reverse); CYP3A4, 5 -GGTGGTGAATGAAACGCTCAG-3 (forward) and 5 -CACCCCTTTGGGAATGAACA-3 (reverse); HEX, 5 -CGCGCTAAATGGAGGAGACT-3 (forward) and 5 -TGGGCAAATCTTGCCTCTG-3 (reverse). Periodic Acid Schiff staining and uptake of indocyaningreen The Periodic Acid Schiff staining system was from Sigma-Aldrich. Cultures were fixed with 4% paraformaldehyde in PBS and then stained according to the manufacturer s instructions. Indocyanin green uptake was performed as previously described. Oil Red O staining For lipid detection, cultures were fixed with 4% paraformaldehyde in PBS and treated with 60% isopropanol for 5 min. Then the isopropanol was removed and Oil Red O working solution (3 parts of 0.5% Oil Red O dissolved in 100% isopropanol diluted with 2 parts water) was added and incubated for 15 min at room temperature. Then the Oil Red O was removed and cultures rinsed with until clear. Animals and transplantation palb-rtta, ptre-upa transgenic mice were generated previously. The transgenic mice were crossed with Rag2 -/- /γc -/- mice. The animals homozygous for the Rag2 -/- /γc -/ trait and hemizygous for the palb-rtta and ptre-upa transgenes were used for transplantation experiments. Differentiated cells or primary human hepatocytes ( cells/animal) were

7 injected into the right lateral liver lobe of newborn mice, which were sacrificed three weeks later. Blood samples were collected and human albumin was quantified in sera using the Human Albumin ELISA Quantitation kit (Bethyl Laboratory). Livers were embedded in OCT compound (Sakura) then frozen in liquid nitrogen. Cryostat sections (10 μm) were stained using an antibody that specifically recognizes human but not mouse KRT18 and albumin to identify human cells. Lentivirus-mediated shrna interference and over-expression For knockdown experiments, lentiviral plasmids containing shrnas were from Sigma-Aldrich. For PROX1-specific and HNF6-specific shrnas, five clones were purchased and the two with the highest knockdown efficiency were used for further experiments. shrna specific to EGFP was used as non-targeted shrna control. For over-expression experiments, lentiviral plasmids carrying full length cdna of human PROX1 and HNF6 (purchased from Origene) were used. The differentiation cultures were infected by lentivirus after the hepatoblast stage (day 11) and characterized by immunofluorescence staining and RT-PCR at the final stage (day 20).