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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

The role of cell differentiation state on hepatitis B virus (HBV) replication has been well demonstrated, whereas how it determines cell susceptibility to HBV entry is far less understood. We previously showed that umbilical cord matrix stem cells (UCMSC) can be differentiated towards hepatocyte-like cells in vitro. In this study we infected undifferentiated (UD-) and differentiated (D-) UCMSCs with HBV and studied the infection kinetics, comparing them to primary human hepatocytes (PHHs). UD-UCMSCs, although permissive to viral binding, had a very limited uptake capacity, whereas D-UCMSCs showed binding and uptake capabilities similar to PHHs. Likewise, asialoglycoprotein receptor (ASGPR) was up-regulated in UCMSCs upon differentiation. In D-UCMSCs, a dose-dependent inhibition of HBV binding and uptake was observed when ASGPR was saturated with known specific ligands. Subsequent viral replication was shown in D-UCMSCs but not in UD-UCMSCs. Susceptibility of UCMSCs to viral replication correlated with the degree of differentiation. Replication efficiency was low compared to PHHs, but was confirmed by (1) a dose-dependent inhibition by specific antiviral treatment using tenofovir; (2) the increase of viral RNAs along time; (3) de novo synthesis of viral proteins; and (4) secretion of infectious viral progeny. Conclusion: UCMSCs become supportive of the entire HBV life cycle upon in vitro hepatic differentiation. Despite low replication efficiency, D-UCMSCs proved to be fully capable of HBV uptake. Overall, UCMSCs are a unique human, easily available, nontransformed, in vitro model of HBV infection that could prove useful to study early infection events and the role of the cell differentiation state on such events. (HEPATOLOGY 2013)

Hepatitis B virus (HBV), as all hepadnaviruses, is hepatotropic and highly species-specific. The reasons for such specificity have not been clarified yet. The role of the cell differentiation state on HBV replication has been well demonstrated,1-3 whereas how it determines cell susceptibility to HBV entry is far less understood.

Although hepatoma cell lines can replicate the virus efficiently after transfection of the viral genome, they are not supportive of HBV entry.4-6 Thus, viral entry seems to be the most important determinant of HBV hepatotropism. Nevertheless, the responsible receptor(s) has not been identified yet.7 Asialoglycoprotein receptor (ASGPR), a hepatocyte-specific lectin, is one of the candidate membrane proteins that have been suggested to play a role in HBV binding and uptake.8-10

Study of early infection events has been limited by the lack of suitable models. Primary human hepatocytes (PHHs) represent the gold standard, but their use is significantly hampered by limited availability, rapid dedifferentiation in vitro, and very high variability between donors.11 Hepatocytes of the tree shrew Tupaia belangeri and the HepaRG cell line have been shown as being infectable by HBV,12-14 but they share limitations of PHHs in terms of accessibility, reproducibility, and low yield of infection.

We previously showed that mesenchymal stem cells (MSCs) from different sources can be differentiated in vitro to hepatocyte-like cells.15-17 Such a potential, together with their self-renewal ability, make MSCs good candidates to study the role of differentiation of HBV-cell interactions.

In the current study we used umbilical cord matrix stem cells (UCMSCs) and showed that they can support the entire HBV life cycle upon hepatogenic differentiation. Although they were shown to replicate the virus with limited efficiency, they proved to be fully capable of HBV uptake. We analyzed the expression of ASGPR by UCMSCs, and its role in viral uptake, as a proof of concept for the use of this easily accessible, nontransformed human model for studies on early HBV infection events.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

A detailed description of all methods used is provided in the Supporting Material.

UCMSC and Human Hepatocytes.

The present study was approved by the local ethical committee. Umbilical cords were obtained from healthy donors after an informed consent was signed. UCMSCs were isolated by collagenase type I digestion of Wharton's jelly according to a well-standardized method we described previously, and cultured in standard conditions.16 Cells were used between passages 4 and 10. PHHs were obtained from the Hepatocytes and Hepatic Stem Cells Bank of Cliniques Saint-Luc (Brussels, Belgium). They were isolated from deceased donors' livers by two-step collagenase perfusion as described,18 and cultured in serum-free Williams' E medium (Invitrogen) supplemented with 100 U/mL penicillin / 100 μg/mL streptomycin (Invitrogen), 10−6 M dexamethasone (Organon), and 10 μg/mL insulin (Lilly Benelux).

Differentiation Procedure.

Hepatic differentiation was induced on UCMSCs (D-UCMSCs) at passages 4-10 according to the described procedure.15, 16

Undifferentiated UCMSCs (UD-UCMSCs) used as controls were cultured in Iscove's modified Dulbecco's medium (IMDM, Invitrogen) supplemented with 1% penicillin/streptomycin and 2% fetal bovine serum (control medium) in standard conditions.

Viral Sources.

Serum from three HBeAg-positive, immunotolerant, treatment-naïve patients was collected after informed consent. A viral load of 1.1 ± 0.6 × 1010 IU/mL was measured. All patients were infected with wildtype HBV of genotype D. As a great variability in infection efficiency was to be expected,11 we attempted to standardize infection conditions using HBV produced in vitro by HepAD38 cells. These cells were cultured as described (detailed description and characterization in the Supporting Material).19 Under such conditions, HepAD38s produced 6.2 ± 2.7 × 108 IU HBV/mL. Such a virus resulted in a wildtype genotype D, subtype ayw, with no genotypic resistance to lamivudine, entecavir, or adefovir identified (Supporting Fig. 2). The virus was concentrated 30 times by polyethylene glycol 6000 (PEG) precipitation, reaching a final concentration of 1.7 × 1010 IU/mL of HBV, and stored at −80°C until use.

HBV Binding Assay.

D-UCMSCs, UD-UCMSCs, and PHHs, cultured in 6-well plates, were incubated with concentrated HBV from HepAD38 in IMDM supplemented with penicillin/streptomycin. Incubation with HBV was carried out for 2 hours at 4°C. Multiplicity of infection (MOI) was calculated assuming that one viral genome equivalent (vge) corresponds to one infectious particle. After 2 hours, as large amounts of viral particles are known to remain nonspecifically bound to cell membrane,20 the cells were extensively washed with cold phosphate-buffered saline (PBS). After the fourth washing, viral DNA in supernatant was <100 vge/mL (Supporting Fig. 4D). Thereafter, DNA was extracted without previous treatment with trypsin to avoid detachment of receptor-bound HBV.

Protease protection assay was carried out after washing by adding 1 mL of 0.25% trypsin to each well and incubation for 10 minutes at 37°C. DNA was then extracted after stopping the reaction and pelleting the cells.

HBV Uptake and Viral Replication Kinetics.

After incubation with HepAD38-derived HBV for 2 hours at 4°C and extensive washing, the cells were moved to a 37°C environment and cultured as described above. DNA was extracted after 1, 4, and 24 hours and after 3, 7, and 10 days. A 10-minute treatment with 0.05% trypsin-EDTA solution, followed by pelleting, was carried out before DNA extraction in order to detach all viral particles still bound to the cell membrane. Conditioned medium was collected at days 1, 3, 7, 10, and 14 and stored at −20°C until use. All experiments were repeated using HBV from patients' sera and no difference in HBV uptake or replication was found (Supporting Fig. 6A,B).

Binding and Uptake Inhibition.

D-UCMSCs were preincubated with either 5 mM EDTA, 1 mg/mL thyroglobulin (with and without EDTA), or 100 μg/mL suramin (with and without EDTA) for 1 hour at 37°C. Such doses of ligands (all from Sigma) have previously proved effective towards ASGPR inhibition in other models.8, 21-23 Cells were then inoculated at an MOI of 103 for 2 hours at 4°C, then extensively washed with cold PBS. DNA was extracted and membrane-bound HBV DNA quantified by quantitative polymerase chain reaction (qPCR).

For uptake inhibition, D-UCMSCs were pretreated with increasing concentrations of D-galactose (0.03-100 μM; Sigma) for 1 hour at 37°C, then inoculated at an MOI of 103, in the presence of the inhibitor, for 4 hours at 37°C. DNA was extracted after extensive washing and trypsin treatment.

Total HBV DNA Quantitation.

We designed a TaqMan assay (RC01; Applied Biosystems ID:AIS07DM) able to specifically amplify a 106-bp region of the precore/core protein gene of genotype D HBV genome (which was common to all our viral sources). All samples were analyzed in triplicate by qPCR with a TaqMan standard 40-cycle amplification program with both annealing and elongation performed at 60°C. Beta-actin was used as the reference gene (Applied Biosystems). The assay proved to have a very low limit of detection (4.5 IU with a Ct <38, hit rate 100% on 12 tests) and a wide dynamic range (up to 3.5 × 1010 IU, R2 = 0.999, PCR efficiency = 98.8%, P < 0.0001), while being highly reproducible and 100% specific for HBV DNA (Supporting Fig. 3). Full details on RC01 assay's in silico analysis, validation with WHO 2nd HBV International Standard, and determination of sensibility and specificity are available in the Supporting Material.

Covalently Closed Circular DNA (cccDNA) Quantitation.

Ten μL of extracted DNA from each sample was digested with 30 IU of Plasmid-Safe DNase (PS-DNase, Epicentre Biotechnologies) in a total volume of 50 μL, for 60 minutes at 37°C. PS-DNase selectively and efficaciously degrades linear DNA or circular single-stranded DNA without affecting cccDNA.24 We used RC01 assay to quantify cccDNA by qPCR. The results were normalized versus β-actin amplification in undigested samples. Detailed evaluation of PS-DNase digestion efficacy and RC01 specificity for cccDNA detection is available in the Supporting Material.

Viral RNA Assessment.

RNA was extracted from D-UCMSCs at days 1, 3, and 7 postinfection. After DNase I treatment and reverse transcription, pregenomic (pg) and precore (preC) RNAs were measured by TaqMan qPCR using the RC01 assay as described above. A sample containing 1 ng plasmid pAM6 (corresponding to 34.6 × 109 IU HBV DNA/mL) was added to each experiment as internal control for efficient DNA digestion and results were excluded if amplification was detected in such a control. Experiments performed to assess specificity of reverse transcription (RT)-qPCR for viral RNAs are described in the Supporting Material.

Viral Replication Inhibition Assay.

D-UCMSCs from three different donors (five experiments) were preincubated with 0.07-2.5 μg/mL tenofovir (phosphonylmethoxypropyladenine [PMPA], Rega Institute, Leuven, Belgium), for 1 hour. They were then inoculated at an MOI of 105 for 4 hours at 37°C, washed, and cultured in differentiation medium supplemented with PMPA over the full course of the experiment. DNA was extracted 7 days postinfection and intracellular HBV DNA was quantified by qPCR. Anti-HBV activity of PMPA was also tested on HepAD38 cells as described.19, 25

Viral RNA (pg and preC) was measured in 2.5 μg/mL PMPA-treated D-UCMSCs at 1, 3, and 7 days postinfection and compared to untreated cells as described above.

Immunofluorescence Analysis.

To assess HBV uptake by the cells studied, and cellular ability to synthesize viral proteins, PHHs, UD- and D-UCMSCs were inoculated at an MOI of 105 for 4 hours at 37°C. After extensive washing they were cultured in standard conditions for 24 hours. After fixation, permeabilization, and preblocking, a mouse monoclonal antibody against HBcAg (NCL-HBcAg-506, Leica) was used (1:100). Staining for ASGPR was obtained on noninfected cells with a rabbit polyclonal antibody (1:400; HPA011954, Sigma). Alexa Fluor 594-labeled antibodies (A11012, A21203, Invitrogen) were used for secondary staining (1:200). DAPI was used to stain the nuclei. Results were analyzed with the Cell Observer SD laser confocal microscope (Carl Zeiss).

Enzyme-Linked Immunosorbent Assay (ELISA) for HBeAg and HBsAg.

Conditioned media from a culture of 150,000 cells were concentrated 10 times by centrifugation at 4,000g for 15 minutes in 10 kDa Amicon Ultra centrifugal filter tubes (Millipore). The resulting samples were analyzed for HBeAg and HBsAg (Monolisa HBeAg/Ab Plus and HBsAg Ultra, Bio-Rad) according to the manufacturer's instructions. Reading of the optical densities at 450/620 nm was carried out with an absorbance reader (Reader 250, BioMérieux).

Western Blot Analysis for ASGPR and HBcAg.

For ASGPR detection, 20 μg of total cellular proteins was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes through semidry transfer. After blocking, a rabbit polyclonal antibody against ASGPR (HPA011954, Sigma) was used for overnight incubation at 4°C (1:5,000). A goat polyclonal anti-α-actin antibody (1:2,000; sc-1616, Santa Cruz Biotechnology) served as internal control. Fluorescent secondary antibodies (Biotium) were used (1:10,000) and the results were read with the Odyssey infrared imaging system (Li-Cor Biosciences).

For HBcAg detection, an immunoprecipitation was performed on lysate from 106 cells (10 days postinfection) using protein A Sepharose beads (GE Healthcare) bound to a rabbit polyclonal anti-HBc antibody (B0586, Dako). After SDS-PAGE separation and transfer, staining was obtained incubating the membrane with a mouse monoclonal antibody against HBcAg (1:2,000; NCL-HBcAg-506, Leica).

Infectivity Assay.

Conditioned medium was collected from D-UCMSCs culture 14 days postinfection. After centrifugation to remove cellular debris, HBV was concentrated by PEG precipitation. Pellets were resuspended in PBS and used to infect PHHs by overnight incubation at 37°C. After extensive washing, PHHs were cultured as described above. DNA was extracted after 24 hours and 7 days to test for viral replication.

Statistical Analysis.

Statistical analysis was performed with PRISM 4 (GraphPad Software). Mann-Whitney U test, Wilcoxon signed-rank test, and one-sample two-tailed t test were used as appropriate. P ≤ 0.05 was considered significant. Values are expressed as mean ± standard deviation or standard error of the mean.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Hepatic Differentiation of UCMSC.

UCMSCs were isolated from Wharton's jelly of six healthy donors. Their identity was confirmed as described by us and others (Supporting Material and Supporting Fig. 1).16 They were differentiated in vitro to hepatocyte-like cells at passages 4 to 10 (Fig. 1A), according to a well-established multistep protocol.15, 16 Quality of differentiation was proven by an increased gene expression of cytochrome P450 3A4 (CYP3A4; P = 0.016), hepatocyte nuclear factor 4-α (HNF4α; P = 0.031), and albumin (P = 0.016), and the exhibition of functions typical of mature hepatocytes, such as CYP3A4 activity (P = 0.031; Fig. 1B,C). Variability in differentiation quality among different donors is shown in Supporting Fig. 1E.

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Figure 1. Hepatogenic differentiation potential of UCMSCs. (A) Morphology of UD-UCMSCs and D-UCMSCs at confluence, before infection experiments. At the end of the differentiation protocol, D-UCMSCs show a polygonal shape with granular cytoplasm. Original magnification 100× and 400×. (B) Increased expression of CYP3A4 (354.7 ± 109.8, n = 6, P = 0.016), albumin (32.2 ± 17.2, n = 6, P = 0.016), and HNF4α (9.34 ± 0.89, n = 5, P = 0.031) mRNAs, in D-UCMSCs compared to UD-UCMSCs (Wilcoxon signed rank test). (C) Increased CYP3A4 activity (luciferin-IPA test) in D-UCMSCs as compared to UD-UCMSCs (n = 5; P = 0.031, Wilcoxon signed rank test).

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HBV Binding to UCMSC Membranes.

To study the effect of differentiation state on HBV-cell interactions, we first assessed viral attachment to the cell membranes. At a temperature below 18°C, endocytosis is inhibited (Supporting Fig. 4A), whereas binding of viral particles to membrane receptors remains active.26 We incubated PHHs, UD-UCMSCs, and D-UCMSCs with HBV at an MOI of 1.0 ± 0.8 × 105, for 2 hours at 4°C. Under these conditions, endocytosis was totally inhibited while cellular viability was not affected (Supporting Fig. 4B,C). After extensive washing (Supporting Fig. 4D), the amount of membrane-bound HBV DNA was similar for PHHs and D-UCMSCs, but lower for UD-UCMSCs (P = 0.052; Fig. 2A).

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Figure 2. D-UCMSCs permit HBV binding and uptake. (A) Binding of HBV on UCMSCs after 2-hour incubation at 4°C compared to PHHs. Results are expressed as vge/cell (n = 6 for UD- and D-UCMSCs, n = 3 for PHHs; P = ns). (B) Protease sensitive binding of HBV to the cell surface: treatment with 0.25% trypsin-EDTA for 10 minutes, at the end of incubation and before DNA extraction, detached 95% of membrane-bound HBV (n = 5 for UD- and D-UCMSCs, n = 3 for PHHs; **P < 0.001 and ***P < 0.0001 as compared to no protease treatment, one-sample two-sided t test). (C) D-UCMSCs were as efficient as PHHs in internalizing membrane-bound HBV after temperature shift to 37°C. Results are expressed as percentage of the internalized (i.e., protease-protected) versus cell membrane-attached (protease-sensitive) HBV DNA after a given incubation period (n = 5 for UD- and D-UCMSCs, n = 3 for PHHs; #P = 0.016 compared to 1 hour postinfection and **P = 0.004 compared to UD-UCMSCs, Mann-Whithney test). (D) An MOI of 100 was needed to achieve at least 1 vge/cell in D-UCMSCs at 24 hours postinfection. No further increase was observed for MOIs >103 (n = 3, P = ns). (E) Immunofluorescence for HBcAg on PHHs, UD-UCMSCs, and D-UCMSCs 24 hours postinfection (confocal laser microscopy, AlexaFluor-594/DAPI staining; original magnification 400×).

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To prove that binding of viral particles on cell membrane was receptor-mediated, after incubation with HBV at 4°C and extensive washing, cells were treated with trypsin before DNA extraction. Protease detached 95% of the viral particles, without any difference between cell types (Fig. 2B), indicating that proteinaceous structures were involved in HBV binding.

Uptake of Membrane-Bound HBV.

To assess whether viral particles attached to membrane receptors could be internalized, after the 2-hour incubation at 4°C and extensive washing cells were moved to a 37°C environment. They were cultured under standard conditions and DNA was extracted after 1, 4, and 24 hours. To make sure to extract only intracellular DNA, trypsin was applied before DNA extraction, in order to detach all particles still bound to the cell membrane.

After 1 hour at 37°C, PHHs, UD-UCMSCs, and D-UCMSCs were able to internalize 4.9 ± 0.7%, 6.3 ± 1.5%, and 5.5 ± 1.3% of membrane-bound HBV, respectively (P = ns; Fig. 2C). The proportion of viral uptake increased at 4 and 24 hours for PHHs (P = ns) and D-UCMSCs (P = 0.016), but remained stable for UD-UCMSCs. HBV uptake after 24 hours was significantly greater in D-UCMSCs than in UD-UCMSCs (P = 0.004). The amount of virus taken up by D-UCMSCs at 24 hours increased with the increase of MOI (Fig. 2D). Little increase was seen for MOI >103, suggesting saturation of the receptor(s).

Viral entry after 24 hours at 37°C was confirmed by immunofluorescence. Both PHHs and D-UCMSCs, but not UD-UCMSCs, showed a positive staining for intracellular HBcAg (Fig. 2E).

ASGPR Is Expressed by UCMSCs and Its Saturation by Competitive Inhibitors Prevents HBV Binding and Uptake.

ASGPR, a Ca2+-dependent hepatocyte-specific lectin,27 was significantly more expressed by D-UCMSCs than by UD-UCMSCs, both at messenger RNA (mRNA) and protein levels (Fig. 3A,B). In this regard, D-UCMSCs resembled PHHs serving as positive controls.

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Figure 3. Role of ASGPR in HBV binding to D-UCMSCs. (A) ASGPR was more strongly expressed by D-UCMSCs than by UD-UCMSCs both at mRNA (n = 6, P = 0.16, Wilcoxon signed rank test; RT-qPCR) and protein (western blot analysis) levels. (B) Immunofluorescence for ASGPR on uninfected PHHs, UD-UCMSCs, and D-UCMSCs (confocal laser microscopy, AlexaFluor-594/DAPI staining; original magnifications 250× and 1,000×). (C) Correlation between ASGPR mRNA expression and D-UCMSCs susceptibility to viral uptake (R2 = 0.924, P = 0.009, n = 5). Uptake capacity is expressed as intracellular HBV DNA (vge/cell) at 24 hours postinfection. ASGPR expression is expressed as fold change as compared to UD-UCMSCs. (D) Incubation of D-UCMSCs before and during HBV infection (MOI 103 for 2 hours at 4°C) with either 5 mM EDTA, 1 mg/mL thyroglobulin (with and without EDTA), or 100 μg/mL suramin (with and without EDTA) led to inhibition of HBV binding (membrane-bound HBV DNA measured by qPCR, n = 3, P < 0.0001, one-sample two-sided t test). (E) HBV uptake by D-UCMSCs was inhibited by D-galactose in a dose-dependent manner (EC50 = 0.2 μM; treatment before and during 4 hours of inoculation with HBV at an MOI of 103). Intracellular HBV DNA levels measured by qPCR are shown (expressed as fold change versus no galactose; n = 3; *P = 0.03, **P < 0.01, ***P < 0.001, one-sample two-sided t test).

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ASGPR has been suggested to play a role in HBV binding and uptake.8-10 HBV uptake by D-UCMSCs was directly correlated with ASGPR mRNA expression (R2 = 0.924, P < 0.01; Fig. 3C). In Fig. 3D we show that HBV binding to D-UCMSCs may be partially inhibited by Ca2+ chelation (21.1 ± 2.5% inhibition) and thyroglobulin addition (77.8 ± 1%), the latter being one known ASGPR-specific ligand. Suramin, which is known to block HBV attachment,22, 23 inhibited 87.4 ± 1% of HBV binding to D-UCMSCs. The addition of increasing concentrations of D-galactose (0.03-100 μM), before and during inoculation of D-UCMSCs, resulted in a dose-dependent inhibition of HBV uptake (up to 79.3 ± 0.7%, P < 0.0001; Fig. 3E). The median effective concentration (EC50) = 0.2 μM (95% confidence interval [CI], 0.17-0.23) was calculated for D-galactose by dose-response analysis (Supporting Fig. 4F).

Cell Susceptibility to HBV Replication.

Total HBV DNA was quantified by qPCR at 3 and 7 days postinfection and compared to the amount of viral DNA present inside the cells at 24 hours postincubation (Fig. 4A). Intracellular HBV DNA levels decreased along time in UD-UCMSCs (P < 0.0001), whereas they increased in PHHs (P = ns) and D-UCMSCs (P = 0.016) at day 3 and 7, suggesting productive viral replication. At 7 days postinfection, intracellular HBV DNA levels did not differ between D-UCMSCs and PHHs, but were significantly higher in D-UCMSCs than in UD-UCMSCs (P < 0.01). A further increase of HBV DNA levels was seen at 10 days postinfection in D-UCMSCs (P = 0.029; Fig. 4B). PHHs were not tested beyond 7 days postinfection to avoid biases due to dedifferentiation.11 In D-UCMSCs, an MOI of at least 100 was needed to yield detectable viral replication (Fig. 4C). Increase of HBV DNA replication intermediates along time was confirmed at Southern blotting on the same samples used for qPCR (Supporting Fig. 5C).

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Figure 4. HBV replication in UCMSCs. (A) Total intracellular HBV DNA at 3 and 7 days postinfection (qPCR), as compared to the amount of HBV DNA internalized by the cells at 24 hours postinfection (represented by the horizontal line; n = 6 for D-UCMSCs, n = 4 for UD-UCMSCs and n = 3 for PHH; *P < 0.05, **P < 0.01, ***P < 0.001 compared to 24 hours, one-sample two-sided t test; #P < 0.05 and ##P < 0.01 compared to UD-UCMSCs; Mann-Whitney test). (B) At 10 days postinfection, intracellular HBV DNA further increased in D-UCMSCs but not in UD-UCMSCs (qPCR, n = 6 for D-UCMSCs and n = 4 for UD-UCMSCs, P = 0.029; Mann-Whitney test). (C) In D-UCMSCs, an MOI of at least 100 was needed to have detectable viral replication (expressed as fold change of intracellular HBV DNA levels at 7 days postinfection versus 24 hours postinfection; n = 3, P = ns). (D) cccDNA at 7 days postinfection, expressed as copies/cell (qPCR, n = 5 for D-UCMSCs, n = 4 for UD-UCMSCs and n = 3 for PHH; P = ns, Mann-Whitney test). (E) Postinfection differentiation assay: differentiation of UD-UCMSCs 24 hours postinfection led to viral replication of the small amount of HBV already internalized (n = 3 for postinfection diff. UCMSCs [black bars], n = 4 for UD-UCMSCs [gray bars]; *P = 0.018 as compared to UD-UCMSCs; one-sample t test). (F) Correlation between differentiation status and cell susceptibility to viral replication. The state of differentiation is expressed as relative activity of CYP3A4 as compared to the average activity in UD-UCMSCs. Ability of the cells to support viral replication is expressed as relative increase of intracellular HBV DNA levels at day 7 postinfection as compared to the virus internalized after 24 hours at 37°C. Both UD- and D-UCMSCs are considered in this analysis (R2 = 0.92, P < 0.0001, n = 9: n = 6 for D-UCMSCs and n = 3 for UD-UCMSCs).

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We subsequently measured cccDNA by qPCR (Fig. 4D). In D-UCMSCs, cccDNA levels increased along time up to 0.03 ± 0.04 copies/cell at 7 days postinfection, corresponding to approximately every 30th cell containing at least one copy of cccDNA. By contrast, 0.7 ± 0.8 copies/per cell were found in PHHs, and no cccDNA was detected in UD-UCMSCs. Controls to prove specificity of cccDNA detection by qPCR are shown in the Supporting Data.

To further confirm the effect of differentiation on cellular susceptibility to viral replication, we infected UD-UCMSCs as described above and induced differentiation after 24 hours. For this postinfection differentiation we used a serum-free medium corresponding to the final step of the differentiation protocol described above. HBV DNA levels in UCMSCs undergoing postinfection differentiation increased along time as compared to UD-UCMSCs (P = 0.018), suggesting replication of the small quantity of HBV (0.22 ± 0.32 vge/cell) internalized by undifferentiated cells (Fig. 4E).

HBV replication efficiency in UCMSCs varied among different donors, and was directly correlated with the quality of differentiation (R2 = 0.92, P < 0.0001; Fig. 4F).

To assess whether known antivirals could inhibit viral replication in D-UCMSCs, the cells were inoculated at an MOI of 105 in the presence of an increasing concentration of PMPA (0-2.5 μg/mL; Fig. 5A,B). A dose-dependent inhibition of HBV replication was shown after 7 days of PMPA treatment (Fig. 5B). EC50 for PMPA was 0.21 μg/mL (95% CI, 0.12-0.39) in D-UCMSCs, as compared to 0.12 μg/mL (95% CI, 0.11-0.14) in HepAD38 (Supporting Fig. 5A).

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Figure 5. (A,B) Inhibition of HBV replication in D-UCMSCs by tenofovir. (A) Intracellular HBV DNA levels 7 days postinfection in D-UCMSCs treated with increasing concentrations of PMPA (vge/cell, n = 5, **P = 0.008; Mann-Whitney test). (B) Dose-response curve for treatment with PMPA on D-UCMSCs (EC50 = 0.21, 95% CI, 0.12-0.39, n = 5). Response was calculated by measuring intracellular HBV DNA 7 days postinfection. (C,D) Transcription of viral RNAs. (C) Viral RNAs (pg and preC) measured in D-UCMSCs by RT-qPCR at 1, 3, and 7 days postinfection (copies/cell; n = 3, P = ns). (D) Viral RNAs measured in D-UCMSCs after 2.5 μg/mL PMPA treatment (RT-qPCR). Results are expressed as fold change versus no treatment (horizontal line).

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Transcription of Viral Genes.

Viral RNAs (pg and preC) were quantified in D-UCMSCs by RT-qPCR. As shown in Fig. 5C, viral RNAs increased in D-UCMSCs along time, reaching 0.103 ± 0.023 copies/cell at day 7 postinfection. Treatment with 2.5 μg/mL PMPA reduced the amount of viral RNAs found in D-UCMSCs by 30%, 81%, and 97% at 1, 3, and 7 days postinfection, respectively (P = ns; Fig. 5D). Specificity of viral RNA quantification by RT-qPCR was carefully assessed (Supporting Material) and confirmed at each experiment.

De Novo Synthesis of Viral Proteins.

We assessed synthesis of viral proteins in UCMSCs by immunofluorescence at day 10 postinfection. A staining for HBcAg was shown in D-UCMSC, whereas it was absent in UD-UCMSCs (Fig. 6A). Secretion of HBsAg and HBeAg was measured by ELISA at different timepoints postinfection. PHHs secreted increasing amounts of HBeAg from day 3 postinfection (Supporting Fig. 5B). To increase sensibility of the technique, we concentrated proteins from conditioned medium by ultrafiltration before ELISA. A significant increase of both viral antigens was detected over time in D-UCMSCs supernatant (P < 0.05; Fig. 6B,C), whereas they remained negative in UD-UCMSCs. Low-level, yet clearly detectable synthesis of viral proteins was confirmed in D-UCMSCs (but not in UD-UCMSCs) by western blotting for HBcAg after immunoprecipitation (Fig. 6D).

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Figure 6. Synthesis of viral proteins and passage of infectious progeny virus. (A) Immunofluorescence for HBcAg in UD-UCMSCs and D-UCMSCs 10 days postinfection (AlexaFluor-594/DAPI staining). Original magnifications: 400× and 1,000× (confocal laser microscopy). (B,C) Synthesis of HBsAg (B) and HBeAg (C) measured in conditioned medium of UD- (dotted line) and D-UCMSCs (solid line) by ELISA after 10× concentration via ultrafiltration. Cutoff was calculated according to the manufacturer's instructions. Values are expressed as optical density/cutoff (OD/CO ≥1 considered positive; n = 4; *P < 0.05 as compared to 7 days postinfection; #P < 0.05 as compared to UD-UCMSCs). (D) Western blot analysis for HBcAg (21 kDa) on UD- and D-UCMSCs 10 days postinfection, after immunoprecipitation. As internal control, IgG heavy chains are shown at 55 kDa (subsequent staining with the same antibody as used for immunoprecipitation). (E) D-UCMSCs proved to produce fully competent viral particles, as HBV secreted in conditioned medium by D-UCMSCs 14 days postinfection was able to infect PHHs. Three independent infection assays were performed using PHHs from the same donor and concentrated HBV from three different D-UCMSC infection experiments (P = 0.06).

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Infectivity Assay.

We assessed the infectivity of viral particles secreted by D-UCMSCs on PHHs. PHHs from one donor were inoculated with D-UCMSCs-derived HBV (three donors, MOI 21.1 ± 26.6) for 16 hours at 37°C. Intracellular HBV DNA levels increased in PHHs at day 7 postinfection as compared to 24 hours postinfection (8 ± 1.8-fold change, P = 0.06; Fig. 6E), suggesting productive viral replication and confirming the ability of D-UCMSCs to synthesize infectious HBV particles, completing the full viral life cycle.

Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

We describe here a new in vitro nontransformed human model of HBV infection. We show that nonliver-derived mesenchymal stem cells (UCMSCs) are turned permissive to the entire HBV life cycle upon in vitro hepatogenic differentiation. None of the few studies conducted on in vitro infection of other MSCs evaluated binding and uptake kinetics.28, 29 We set up infection conditions in order to analyze the different steps of the viral cycle and demonstrated that, although replication efficiency downstream of viral entry was quite low, HBV uptake was fully supported by D-UCMSCs and comparable to PHHs. To our knowledge, this is the first human, nontransformed in vitro model, other than primary hepatocytes, to be fully capable of HBV uptake.

UCMSCs are widely characterized MSCs that are easy to obtain and to expand in culture. As we previously demonstrated, UCMSCs are capable of exhibiting many of the markers and functions typical of mature hepatocytes.16 Nevertheless, we observed variability in the quality of differentiation among donors. As shown by the direct correlation between CYP3A4 activity (a surrogate marker of differentiation state) and susceptibility to viral replication, quality of differentiation seems to be a key factor influencing efficiency of infection in D-UCMSCs. CYP3A4 activity could therefore be used as a marker to predict suitability of each UCMSCs population for infection studies.

The understanding of the viral life cycle has been hampered by the extremely restrictive tropism of HBV. PHHs are the preferred tissue culture model, but they are difficult to obtain, maintain in culture, and infect in vitro.11 Primary Tupaia hepatocytes (PTH) are an interesting alternative.12, 13 Unfortunately, it is well known that susceptibility of primary hepatocytes to HBV infection is rapidly lost during prolonged culture.11 Such a loss in susceptibility has been correlated to the dedifferentiation process to which hepatocytes rapidly undergo in vitro.30

It has been shown that transcription of HBV genes and subsequent viral replication is dependent upon the degree of differentiation of the host cell.1 Liver-enriched transcription factors such as HNF4α, HNF1α, and HNF3β have been shown to have a central role in regulating viral promoters and enhancers.2, 3 Here we show that UCMSCs express HNF4α mRNA upon differentiation, but at a much lower level than PHHs, which could account for the observed lower intracellular replication efficiency.

The role of the differentiation state on cell susceptibility to HBV entry is far less understood. HBV binding and uptake seem to be the most important determinants of HBV hepatotropism. Although many different hepatoma-derived cell lines have been demonstrated to be capable of viral replication after transfection of the viral genome,4-6 very few in vitro models, besides PHHs, permit viral uptake.12-14 D-UCMSCs susceptibility to HBV uptake therefore makes them a highly promising model.

HBV binding and entry into susceptible cells is a multistep process which is poorly understood. The initial attachment of the virus to the cell membrane is believed to be mediated by heparan sulfate proteoglycans,23 and does not seem to be specific for susceptible cells.26 A more specific binding to one or multiple receptors, expressed mainly on hepatocytes, and localized on the basolateral membrane, is probably responsible for the restricted HBV host range.31 Among the many candidate receptors, ASGPR is the only one that is strictly hepatocyte-specific. It is a lectin expressed on the sinusoidal and basolateral hepatocellular membrane, which is responsible for the uptake of desialylated glycoproteins.27 Basma et al.32 showed that ASGPR is up-regulated in embryonic stem cells upon hepatic differentiation. Different studies have proved it to be necessary for HBV binding and uptake.8-10 Here we show that ASGPR is up-regulated in UCMSCs upon differentiation. We also show a dose-dependent inhibition of HBV binding and uptake when ASGPR is saturated with known specific ligands. Although further verification would be necessary to definitely prove the role of this receptor, these experiments are a proof of concept that UCMSCs may be a suitable model to study early infection events.

HBV is highly infectious in vivo, but only a small proportion of the cells are infected in vitro.33 PTH and HepaRG share the same disadvantages of PHHs in terms of low replication efficiency and high MOI needed to infect a reasonable proportion of cells.12-14 UCMSCs were even less efficient than PHHs in replicating HBV, showed a low-level protein synthesis, and a high MOI was indeed needed to achieve a productive infection. Nevertheless, viral entry was as efficient as in PHHs.

As our aim was to create an in vitro model as “physiological” as possible, and not to maximize infection efficiency, we decided to avoid the use of all adjuvant molecules (such as dimethyl sulfoxide or polyethylene glycol) that could cause possible experimental artifacts. Improvement of the quality of differentiation would be needed to improve infection efficiency of this model.

Taken together, these data show that UCMSCs are a unique human, easily available, nontransformed, in vitro model of HBV infection. Such cells could prove useful to study early infection events and the role of the cell differentiation state on such events.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

We thank Dr. Patrick Van Der Smissen (de Duve Institute, Cellular Biology Unit), Mrs. Nawal Jazouli, Mrs. Floriane André, Mr. Joachim Ravau, and Mr. Jonathan Evraerts (Pediatric Hepatology and Cell Therapy Lab) for technical assistance.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
HEP_26006_sm_SuppFig1.tif10240KSupporting Information Figure 1.
HEP_26006_sm_SuppFig2.tif2556KSupporting Information Figure 2.
HEP_26006_sm_SuppFig3.tif881KSupporting Information Figure 3.
HEP_26006_sm_SuppFig4.tif2041KSupporting Information Figure 4.
HEP_26006_sm_SuppFig5.tif5077KSupporting Information Figure 5.
HEP_26006_sm_SuppFig6.tif3918KSupporting Information Figure 6.
HEP_26006_sm_SuppInfo.doc122KSupporting Information

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