Isolation and culture of normal and mutated human liver stem cells

A liver specimen from a 5-year-old citrullinemia type I patient undergoing liver transplant was obtained under written informed consent from the parents. The tissue was provided anonymously according to the local tissue banking protocol of discarded tissues (# 80911) approved by the University of Torino Ethics Board (Comitato Bioetica di Ateneo).

Hepatocytes were isolated from the liver specimen (5–20 g) by collagenase digestion following the same protocol as for the isolation of normal HLSCs as described previously [5]. Briefly, isolated liver tissues were digested in liver digest medium (Invitrogen, Carlsbad, CA, USA) at 37 °C. The cell suspension obtained was filtered through a sterile 100-μm nylon mesh, centrifuged at 3000 g for 5 min and subsequently washed in cold wash medium (Invitrogen). The hepatocytes obtained were cultured in a medium composed of three parts α-minimum essential medium and one part endothelial cell basal medium-1 (3:1; Lonza, Basel, Switzerland) supplemented with l-glutamine (5 mM), penicillin (50 IU/ml), streptomycin (50 g/ml) (all from Sigma-Aldrich, St. Louis, MO, USA), and 10% fetal calf serum (FCS; Invitrogen). After a few days of culture ASS1-HLSC clones were expanded and subsequently frozen. Wild-type HLSCs were isolated from cryopreserved normal human hepatocytes (hH) obtained from Cambrex Bio Science, Verviers S.p.r.l. (Verviers, Belgium). hH were also used as normal control of mature hepatocytes.

Isolation of HLSC-derived EVs (HLSC-EVs)

Briefly, 2 × 106 HLSCs and shRNA ASS1 knockdown HLSCs (HLSC shRNA-ASS1) were cultured in serum-free Roswell Park Memorial Institute (RPMI) medium for 18 h at 37 °C. The supernatant was centrifuged for 20 min at 3000 g to remove cell debris and apoptotic bodies. This was followed by a two-step ultracentrifugation protocol whereby the supernatant was firstly centrifuged at 10,000 g (10 K) for 1 h at 4 °C to pellet the 10 K EV fraction and then a second ultracentrifugation at 100,000 g (100 K) for 1 h at 4 °C to isolate the 100 K EV fraction (Beckman Coulter Optima L-90 K, Fullerton, CA, USA) [19]. Both the 10 K and 100 K fractions were resuspended in RPMI supplemented with 1% dimethyl sulfoxide (DMSO) and frozen at –80 °C for later use. The viability of HLSCs at the time of EV collection was 97–99% as determined by trypan blue exclusion.

As suggested by Kowal et al. [20], an iodixanol (Optiprep from Sigma-Aldrich) floating separation protocol was used to further purify the EV fractions from free floating contaminating proteins and RNAs [21]. Briefly, the pellets of EVs obtained after differential ultracentrifugation were directly resuspended in 500 μl 60% iodixanol (Optiprep from Sigma-Aldrich) mixed with 0.25 M sucrose and transferred to ultracentrifuge tubes (10.4-ml polycarbonate centrifuge bottles; Beckman Instruments). Subsequent gradients of iodixanol at 30%, 15%, and 5% were then layered on top of the initial 60% EV-iodixanol/sucrose preparation and the final volume adjusted to 10 ml by topping up with saline solution. The tubes were then ultracentrifuged at 350,000 g for 1 h, 4 °C, without braking in an Optima L-100 K ultracentrifuge (Beckman Coulter) equipped with type 90Ti rotor. Following ultracentrifugation, the 15%, 30%, and 60% fractions were recovered, diluted with phosphate-buffered saline (PBS), and ultracentrifuged again at 100,000 g for 1 h. The pellet obtained was resuspended in RPMI supplemented with 1% DMSO and either used fresh or stored for subsequent studies under –80 °C conditions. No difference in biological activity was observed between fresh and stored EVs.

In order to study the internalization of EVs by fluorescent microscopy, EVs were labeled with 1 μM Dil dye (Thermo Fisher Scientific, Waltham, MA,USA) as described previously [18]. Briefly, purified EVs were resuspended in PBS supplemented with 1 μM Dil dye and ultracentrifuged at 100,000 g for 1 h at 4 °C. Following labeling, the EVs were washed once more with PBS by ultracentrifugation as mentioned above. The pellet obtained was then resuspended in RPMI with 1% DMSO and frozen for subsequent studies.

Concentration and size distribution of EVs were determined by the Nanosight LM10 system (NanoSight, Wiltshire, UK). Briefly, EV preparations were diluted (1:200) in sterile saline solution and analyzed by the Nanoparticle Analysis System using the NTA 1.4 Analytical Software [21].

Flow cytometric analysis

Cytofluorometric analysis was performed as described previously [4]. The antibodies used (conjugated with phycoerythrin (PE) or fluorescein isothiocyanate (FITC)) are as follows: anti-CD45, anti-Albumin, anti-CD90, anti-CD73, anti-CD105, anti-CD29, anti-CD31, anti-KDR, and anti-VE-Cadherin (Dako Denmark A/S, Copenhagen, Denmark). All incubations with antibodies were performed in 100 μl PBS containing 0.1% bovine serum albumin (BSA) at 4 °C. The cells were washed twice between incubations and analyzed (10,000 cells/sample) on a BD FACSCalibur cytometer (BD Biosciences Pharmingen, San Jose, CA, USA). For detection of albumin, cells were permeabilized and labeled with anti-albumin monoclonal antibody (R&D Systems, Abington, UK) followed by PE-conjugated anti-mouse IgG secondary antibody. All samples were gated on the basis of negative controls, and compensated appropriately prior to analyses. Population percentages and numbers were generated for gated populations from each experiment using Cell Quest software (BD Biosciences Pharmingen).

In vitro osteogenic, adipogenic, and endothelial differentiation

Osteogenic, adipogenic, and endothelial differentiation of ASS1-HLSCs was performed as described previously [4]. Briefly, ASS1-HLSCs were cultured in osteogenesis induction medium (Lonza) for 3 weeks (replenished twice per week for 3 weeks). Differentiation was evaluated by fixing cells with 4% paraformaldehyde for 20 min and then staining with alizarin red, pH 4.1 (Sigma-Aldrich), at room temperature. For adipogenic differentiation, cells were cultured in adipogenesis induction medium (Lonza), followed by adipogenic maintenance medium (Lonza). To evaluate the differentiation, cells were fixed with 4% paraformaldehyde for 20 min at room temperature then stained with 0.5% oil red O (Sigma-Aldrich) in methanol (Sigma-Aldrich). Endothelial cell differentiation was obtained by culturing ASS1-HLSCs in EBM (Lonza) with vascular endothelial growth factor (10 ng/ml; Sigma-Aldrich) for 10 days. Following culture, endothelial differentiation was evaluated by flow cytometric analyses of endothelial markers as described previously [4].

Immunofluorescence analysis

Indirect immunofluorescence of cells was performed as described previously [4]. Briefly, ASS1-HLSCs cultured on chamber slides (Nalge Nunc International, Rochester, NY, USA) were fixed with 4% paraformaldehyde and/or permeabilized (only for intracellular markers) with 0.1% Triton X-100 buffer. Cells were then labeled with the following monoclonal antibodies: anti-albumin (R&D Systems), anti-nestin (BD Biosciences Pharmingen), anti-α-fetoprotein (αFP; R&D Systems), anti-nanog (Abcam, Cambridge, MA), anti-Sox2 (Abcam), anti-vimentin (Sigma-Aldrich), anti-cytokeratin 8 (CK8), anti-CK19, anti-Von Willebrand factor (all from Sigma-Aldrich), and anti-ASS1 (Thermo Fisher Scientific). Alexa Fluor 488 anti-rabbit IgG and Texas Red anti-mouse IgG (Thermo Fisher Scientific) were used as secondary antibodies and Hoechst 33258 dye (Sigma-Aldrich) was applied for nuclear staining. Labeling of cells with only secondary antibodies or substitution with nonimmune rabbit, rat, or mouse IgGs served as controls. Slides were analyzed by confocal microscopy using a Zeiss LSM 5 Pascal Model Confocal Microscope (Carl Zeiss International, Jena, Germany).

In vitro culture in RCCS and urea production

In order to differentiate HLSCs and ASS1-HLSCs into functional hepatocytes, cells were cultured in microgravity conditions using the Rotary Cell Culture System (RCCS; Synthecon Incorporated, Houston, TX, USA) as described previously [4]. Briefly, cells (250,000/ml) were resuspended in medium consisting of 75% Dulbecco’s modified Eagle’s medium (DMEM; Mediatech, Herndon, VA, USA), and 25% MCDB 201 (Sigma-Aldrich) supplemented with 2% fetal bovine serum (FBS), linoleic acid (Sigma-Aldrich), l-ascorbic acid (Sigma-Aldrich), insulin-transferrin-selenium (ITS; Sigma-Aldrich), penicillin (100 μg/ml), streptomycin (100 μg/ml), and 10 ng/ml of hepatocyte growth factor and fibroblast growth factor 4; 10 ml of the cell suspension was then loaded into circular RCCS vessels and rotated about their own axis at 8–10 rotations per minute under 37 °C conditions. After 4 days of culture, the supernatant was collected, centrifuged at 300 g for 5 min and stored at –20 °C. Levels of urea in the supernatants were then evaluated using the blood urea nitrogen (BUN) colorimetric detection kit as per the manufacturer’s protocol (Arbor assays, MI, USA). For selective RCCS experiments, cells were cultured in the presence of HLSC-EVs with or without α-methyl-dl-aspartic acid (MDLA), a specific inhibitor of ASS1 enzyme. Treatment of ASS1-HLSCs with ASS1 shRNA EVs or fibroblast EVs (hFib EV) served as controls.

Western blot analysis

The level of ASS1 enzyme in cells was determined by Western blot analyses. Briefly, cells were lysed on ice for 30 min in radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl PH 7.4, 150 mM NaCl, 1% NP-40, 0.1% SDS; Sigma-Aldrich) supplemented with the protease inhibitors phenylmethylsulfonyl fluoride (PMSF) (1 mM), leupeptin (10 μg/ml), and aprotinin (100 units/ml). Proteins from EVs were isolated using the all-in-one purification kit (Norgen Biotek Corp, TO, Canada) according to the manufacturer’s protocol, and the concentrations determined by the Bradford method.

Protein samples at a concentration of 10–30 μg were separated in 8% or 4–15% gradient SDS-PAGE gels under reducing conditions and electroblotted onto 0.2-mm nitrocellulose membranes (GE Healthcare Life Sciences, MA, USA). The membranes were blocked in Tris-buffered saline-Tween (TBS-T; 25 mM Tris, pH 8.0, 150 mM NaCl, and 0.05% Tween-20) containing 5% (w/v) nonfat dried milk for 1 h. After blocking, membranes were probed overnight with mouse anti-ASS1 (BD Transduction Laboratories), mouse anti-CD63, rabbit anti-tubulin, and goat anti-actin (Santa Cruz Biotechnology, CA, USA). After extensive washings with TBS-T, the blots were incubated with appropriate peroxidase conjugated secondary antibodies (goat anti-mouse, goat anti-rabbit, and mouse anti-goat IgG; Pierce, Thermo Fisher Scientific) for 1 h at room temperature. Following incubation, the membranes were washed extensively with TBS-T, probed with enhanced Super Signal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific), and detected by the Chemidoc system (Bio-rad, CA, USA).

Immunoprecipitation and enzymatic activity of ASS1

Cells were lysed in nondenaturing lysis buffer (137 mM NaCl, 2 mM EDTA, 20 mM Tris–HCl, pH 7.5, 10% glycerol, 1% Nonidet P-40, and protease inhibitor cocktail) and centrifuged at 300 g for 15 min at 4 °C. The supernatants were collected and quantified for total protein concentration using the Bio-Rad protein assay method.

Immunoprecipitation of ASS1 was performed using Dynabeads Protein G (Thermo Fisher Scientific). Briefly, the beads were incubated with 10 μg ASS1-antibody (Thermo Fisher Scientific) per mg of total protein for 1.5 h at 4 °C to form complexes. The bead/ASS1-antibody complexes were washed with PBS-0.02% tween and incubated with cell lysates (1 mg total protein) overnight at 4 °C under rotation. After three washes with PBS-0.02% tween, ASS1 proteins were eluted by adding 20 μl 50 mM glycine at pH 2.8. In order to prevent the loss of the tridimensional structure of ASS1, 200 μl TRIS-HCL 20 mM pH 7.8 was added.

Eluted proteins were then evaluated for ASS1 activity using a modified version of the assay described by Lakhal-Naouar et al. [22]. Briefly, eluted protein samples (12.5 μl per reaction) were resuspended in reaction buffer (20 mM Tris-HCl, pH 7.8, 4 mM ATP, 4 mM citrulline, 4 mM aspartate, 6 mM MgCl2, 20 mM KCl, and 0.2 units of pyrophosphatase) to a final volume of 20 μl. The reaction samples were then transferred to 96-well microtiter plates in duplicates and incubated for 30 min at 37 °C. Following incubation the reaction was stopped by adding 20 μl malachite green reagent (Malachite Green Phosphate Assay Kit, Bioassay System, CA, USA). Reactions without citrulline and aspartate substrates were prepared to serve as controls. Phosphate accumulation was determined at 655 nm by spectrophotometry, and the concentration interpolated from a standard curve of inorganic phosphate (Pi). Due to the spontaneous release of Pi in the absence of substrates, the final concentration of Pi from affinity-purified proteins and/or rat liver extract (RLE; served as positive control for the reaction) were determined by subtracting the mean concentration of Pi obtained in the absence of substrates from the mean concentration of Pi obtained in the presence of substrates. The specific ASS1 activity was determined using the following formula: nanomoles of phosphates released/mg protein/30 min [22]. In selected experiments, ASS1-siRNA transfected ASS1-HLSC were used.

RNA extraction and qRT-PCR

Total RNA from cells was extracted using TRIzol reagent (Thermo Fisher Scientific) and total RNA from HLSC-EVs was isolated using the all-in-one purification kit (Norgen Biotek Corp.) according to the manufacturer’s protocol. The quantity and quality of isolated RNAs were determined using the Nanodrop ND-1000 spectrophotometer (Thermo Fisher Scientific). cDNA was then synthesized using the High Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific) as per the manufacturer’s protocol. cDNA at a concentration of 2 ng was used per sample per quantitative real-time polymerase chain reaction (qRT-PCR) reaction in triplicate using a 96-well StepOne Real-Time System (Thermo Fisher Scientific). Wells containing only primers were cycled in parallel with each run and served as negative cDNA controls. For mRNA expression in cells, results were analyzed using the RQ method with GUSB gene as an internal control and for HLSC-EVs, the 2

–∆Ct

method was adopted using S18 gene as internal control. Primers and probes were designed using the Universal ProbeLibrary Assay Design Center software (www. lifescience.roche.com). Primers used to amplify ASS1 mRNAs are summarized in Table 

1

.

Table 1

Primers used in qRT-PCR to evaluate ASS1 mRNA expression

hASS1-Iso1

F1 TGT GAA AAC AGA TTC CACG C

R1 CCA ATG TTG GCC AGA TAG GC

hASS1-Iso2

F1 ACG CTA TGT CCA GCA AAG GC

R1 CCA ATG TTG GCC AGA TAG GC

hASS1-Exon 14 (Primer 1)

F AAGTCCCAGGAGCGAGTGG

R GTGGGGCACCTACCTCACC

hASS1-Exon 15 (Primer 2)

F GCCAGGCTGAGCTGACAAG

R ACCTGGAGGCTCTGAAGGC

ASS1 silencing

A set of five pLKO.1 HIV-based lentiviral vectors targeting the human ASS1 gene were purchased (TRCN0000045553, TRCN0000045554, TRCN0000045555, TRCN0000045556, TRCN0000045557; Dharmacon RNAi Consortium (TRC) Lentiviral shRNA). shRNAs were tested on HLSCs to evaluate the silencing efficiency and the most efficient and stable shRNA was used to specifically downregulate ASS1 expression in HLSCs. The lentiviruses pLKO.1-scr (expressing a “scramble” shRNA as control) and pLKO.1-ASS1 (expressing the shRNA specific for ASS1) were produced in HEK293FT cells using a lentiviral packaging system after which HLSCs were infected with the lentiviruses in the presence of Sequa-brene (Sigma-Aldrich). Following infection, cells were selected with 5 μg/ml puromycin. The efficacy and stability of silencing in selected cells was evaluated by qRT-PCR.

siRNA transfection

ASS-siRNA and nonsilencing siRNA were purchased from Thermo Fisher Scientific. Transient transfection of ASS-siRNA was performed using Lipofectamine RNAiMAX transfection reagent (Invitrogen) as per the manufacturer’s protocol. ASS1-HLSCs (500,000 cells per well) were seeded in a petri dish (10-cm diameter) and cultured for 24 h. The complete culture medium was replaced with antibiotic-free medium and transfected with 300 pM ASS-siRNA or nonsilencing RNA for 3 days in the presence of Lipofectamine RNAiMAX. Before the addition in cell cultures, siRNAs were incubated with lipofectamine for 20 min at room temperature in Opti-Mem medium (Invitrogen). Preliminary experiments using FITC-siRNA (Qiagen, Valencia, CA, USA) indicated that the peak of expression was 3 days after transfection. After 3 days of transfection, ASS1-HLSCs were detached with trypsin and 3.0 × 106 cells were cultured in RCCSs in the presence of 1 × 1010 EV-HLSCs. The effective ASS-siRNA transfection was evaluated in ASS1-HLSCs by RT-PCR. ASS enzymatic activity was measured after 4 days of culture under RCCS culture conditions.

DNA mutation analysis

Genomic DNA was extracted using DNeasy Blood & Tissue Kit (Qiagen) according to the manufacturer’s instructions. The quality and quantity of extracted DNA was determined using the Nanodrop ND-1000 spectrophotometer (NanoDrop Technology).

Two regions of the human ASS1 gene located in exons 14 and 15 were amplified by PCR. These regions comprise of the following codon mutations: R363W (exon 14) and G390R (exon 15). The primers were developed (Table 

2

) based on the GenBank reference sequence (accession no. NC_000009.11), using Primer Express 3.0 software (Thermo Fisher Scientific). PCR was then performed in a final volume of 50 μl, containing 10× PCR buffer, 0.05 units/μl JumpStart Taq polymerase, 10 mM deoxynucleotide triphosphates mix, 150 ng genomic DNA, and 500 nM primers. Cycling conditions were as follows: 1 min at 94 °C, 35 cycles at: 94 °C for 30 s, 62 °C for 30 s, and 72 °C for 60 s, followed by 1 min at 72 °C. At the end of the reaction, 10 μl PCR-amplified DNA samples were analyzed using 1% agarose gel.

Table 2

SNaPshot primers

R363W

T20GGTGTACATCCTCGGC

G390R

T30GCCAACTGATGCCACC

To evaluate the presence of DNA in EVs derived from HLSCs, DNA was extracted and analyzed by PCR. Briefly, 14 different preparations of EVs obtained from HLSCs were pooled together by ultracentrifugation at 100,000 g for 2 h at 4 °C. The supernatant was discarded and lysis buffer was added directly to the EV pellet and processed as per the manufacturer’s protocol to isolate the DNA (DNeasy Blood & Tissue Kit, Qiagen). Exons 14 and 15 of the hASS1 gene were amplified using PCR as described above and amplicons were analyzed using 1% agarose gel.

PCR products from ASS1-HLSCs and ASS1-HLSCs treated with HLSC-EVs were analyzed for mutations (c.1087C > T and c.168G > A) using the ABI PRISM SNaPshot Multiplex Kit (Thermo Fisher Scientific), according to the protocol supplied by the manufacturer (normal HLSCs served as control). The SNaPshot method is based on the dideoxy single-base extension of unlabeled oligonucleotide primers. For each of the two mutations analyzed above, a primer annealing adjacent to the potentially mutant nucleotide was developed (Table 2). Briefly, the SNaPshot reaction was performed in a volume of 10 μl, containing 3 μl PCR product, 5 μl ready reaction mix, 1× sequencing buffer, and SNaPshot primers concentrated at 0.02 pmol/μl. At the end of the reaction, the labeled products were first treated with shrimp alkaline phosphatase (SAP; USB Corporation) to remove excess dideoxynucleotide triphosphate and then separated on 36-cm-long capillaries by performing a 25-min run in an automatic sequencer (ABI PRISM 3500 Genetic Analyzer, Thermo Fisher Scientific). GeneScan Analysis Software version 3.7 (Thermo Fisher Scientific) was used for data analysis. For each sample, the intensity of the fluorescent peak was measured.

Statistical analysis

Results are expressed as mean ± standard deviation (SD). Statistical analysis was performed using the Student’s t test and analysis of variance (ANOVA) with Newmann-Keuls test or ANOVA with Dunnet’s multicomparison tests where appropriate. A p value <0.05 was considered significant.