All used foamy vector (FV) plasmids were derived from the plasmid MD9 (Heinkelein et al., 2002; Peters et al., 2005; Wiktorowicz et al., 2009) that expresses the enhanced green fluorescent protein (EGFP) markergene driven by the spleen focus forming virus (SFFV)-U3 promoter (Figure 1A). The FV plasmids NA1 and KG84 (Figures 1A, 2A) were already described earlier (Gartner et al., 2009; Armbruster et al., 2014). For cloning of the FV vector plasmid construct NA4 that expresses EGFP via an internal ribosomal entry site (IRES) of the encephalomyocarditis virus driven by the human elongation factor 1α (EF1α) promoter (Uetsuki et al., 1989), EF1α was PCR amplified with specific primers using the pEF-GW-51-lacZ plasmid as PCR template (Gateway Vektor System, Invitrogen) and inserted into the pBF014 plasmid via the AfeI and AscI restriction site (Armbruster et al., 2014) (Figure 1A). The plasmid JK1 was derived from a pTW01 based FV plasmid, namely pTW22, coding for mCherry with a fused 2A cleavage site and EGFP (Ryan et al., 1991; Wiktorowicz et al., 2009). The EGFP was replaced with the PCR amplified human IL1RA cDNA, which was inserted via RsrII and SalI (Figure 2A). The structure of each plasmid was verified by restriction mapping and sequencing prior to use. FV vectors were abbreviated with the respective promoter and insert (followed by the name of the corresponding vector plasmid), namely FV.CMV-EGFP (KG48), FV.U3-EGFP (MD9), FV.EF1α-EGFP (NA4), FV.U3-IL1RA-EGFP (NA1), FV.U3-mcherry-IL1RA (JK1) (illustrated in Figures 1A, 2A).

Viral vector stocks were generated by transient transfection of HEK 293T cells as descriebd earlier (Stange et al., 2005; Wiktorowicz et al., 2009; Armbruster et al., 2014). Briefly, 8 × 10⁶ cells were seeded on 10 cm-dishes and were transfected the next day using 20 μg of total plasmid DNA with the following ratio 10:5:1:1 of vector plasmid and packaging plasmids (pCZIgag2, pCZIpol, and pCZ-HFVenvEM002) using polyethylenimine (Polysciences) (Stange et al., 2005). MOCK controls were also performed, using a transfection mix lacking the env plasmid and empty pcDNA to adjust the DNA amount. One day after transfection cellular transcription was induced by addition of 10 mM Na-butyrate for 8 h. After 2 days the supernatants were harvested, passed through a 0.45-μm filter (Millipore) and stored in aliquots at –80°C. Vectors were optionally concentrated to higher titers by centrifugation (polyallomer tubes, 12000 g, 4°C, 2 h). The infectious titer of the FVV preparations were determined using vector dilutions and an immunofluorescence assay as previously described (Peters et al., 2008). Vector preparations were adapted to 3.5 × 10⁷ infectious particles/ml and stored at –80°C. (Peters et al., 2008). FVV and transduced cells were handled in the laboratories of the Department of Virology, University of Wuerzburg under appropriate biosafety level 2 conditions according to German law (Gentechniksicherheitsverordnung-GenTSV).

The vector-containing supernatant was assayed functionally by transfer to 1 × 10⁴ cells by using different doses of infectious virus, as given per multiplicities of infection (MOI) in the respective experiments. For characterization studies the expression of EGFP was monitored by fluorescence-activated cell sorting (FACS) using a FACSCalibur flow cytometer (Beckton Dickinson) if not otherwise mentioned 72 h after transduction. Levels of cell culture infectious dose 50 (CCID₅₀) were determined using the GraphPad prism 4.0 software (GraphPad Software). The vector transfer assays were done at least three times with different plasmid preparations. As recipient cells the human fibroblastic cell line HT1080, the human TERT4 MSC line and primary human MSCs were used (Abdallah et al., 2005). Primary MSCs were obtained from bone marrow of several human donors undergoing total hip replacement surgery after informed consent and as approved by the by institutional review board of Wuerzburg University. MSCs were isolated by adherence of cells harvested from the patient’s spongiosa to plastic and maintained as described previously (Nöth et al., 2002).

For evaluation of IL1RA transgene expressions conditioned media from cell cultures were collected over a 24-h period and stored at –20°C. The human IL1RA concentrations were measured by ELISA (DuoSet Elisa Development Quantikine kit, R&D Systems). The minimum detectable dose of the human IL1RA ELISA is 6.26 pg/ml according to the manufacturer. All measurements were performed in triplicates.

Primary MSCs and the mesenchymal TERT4 cells were transduced with FVVs at 1000 MOI in T-125 flasks at ∼40% confluency to obtain transduction efficiencies of around 50%, which were confirmed by detection of green fluorescence. Three days after transduction EGFP+ cells were sorted using a FACSDiVa (Beckton Dickinson). Selection efficiency was determined to be approximately 99 %. After one week, expansion sorted primary MSCs and mesenchymal TERT4 cells were trypsinized and placed in aggregate cultures as described earlier (Johnstone et al., 1998; Penick et al., 2005). Briefly, cells were distributed in 15 ml polypropylene tubes (Falcon) at a concentration of 3 × 10⁵ cells/pellet in 500 μl medium [serum-free DMEM, 37.5 mg/ml ascorbate, 1 mM pyruvate, 10^-7 M dexamethasone, 1% ITS (insulin, transferrin, and selenous acid containing culture supplement), all Sigma] to promote aggregate formation. To induce chondrogenesis 10 ng/ml recombinant TGFβ1 protein (R&D Systems) was added. Further, aggregates were additionally supplemented with 5 ng/ml IL1β (R&D Sytems) to inhibit chondrogenesis. The aggregates were cultured at 37°C, 5% CO2 and formed spherical pellets within 24 h. Changes of media were performed every 2 to 3 days. The aggregates were harvested after 21 days for further analysis. At least three different aggregates per group and bone marrow preparations from three different preparations were analyzed if not otherwise mentioned. MSC passages for aggregate cultures ranged from passage 4-6.

Cell proliferation in aggregates was assessed by quantitative detection of adenosine 5′-triphosphate (ATP), which correlates with the number of viable cells present, using the CellTiter-Glo^® Luminescent Cell Viability Assay (Promega) according to the manufacturer’s instructions. Briefly, pellets were homogenized mechanically using a pellet pestle and mixed with 100 μl of CellTiter-Glo^® reagent (CellTiter-Glo^® substrate + CellTiter-Glo^® buffer). After incubation for 10 minutes at room temperature luminescence was measured using a plate-reading luminometer.

For analysis of glycosaminoglycan (GAG) content, aggregates were washed with phosphate buffered saline (PBS), digested with 200 μl of papain digest solution (1 μg/ml, Sigma), and incubated for 16 hours at 65°C. Samples were stored at –20°C. Total GAG content was measured by reaction with 1,9-dimethylmethylene blue using the Blyscan^TM Sulfated Glycosaminoglycan Assay (Biocolor Ltd) as directed by the supplier. For normalization, DNA content of aggregates was also determined fluorometrically using the Quant-iT^TM PicoGreen^® kit as directed by the supplier (Invitrogen).

For histological analyses, aggregates were fixed in 4% paraformaldehyde for one hour afterward dehydrated in graded alcohols, embedded in paraffin and sectioned to 5 μm. Representative sections were stained using haematoxylin and eosin (H&E) for evaluation of cellularity and alcian blue (Sigma) for the detection of matrix proteoglycan.

For immunohistochemical analyses sections were washed for 20 min in Tris-buffered saline (TBS) and incubated in 5% bovine serum albumin (BSA) (Sigma). Following washing in TBS, sections were pre-digested with pepsin at 1 mg/ml in Tris–HCl (pH 2.0) for 15 min at room temperature. Sections were then incubated overnight at 4°C with a monoclonal anti-COL II primary antibody (diluted in 0.5% BSA, Acris Antibodies GmbH) for detection of collagen type II (Collagen II). Immunostaining was visualized by treatment with peroxidase-conjugated antibodies (Dako) followed by diaminobenzidine staining (DAB kit; Sigma). The slides were finally counterstained with hemalaun (Merck).

Analysis of viral protein expression was done essentially as described previously (Peters et al., 2005). In brief, cell lysates were prepared using lysis buffer (RIPA plus protease inhibitors). Viral proteins were probed with anti-Gag (Heinkelein et al., 2002), anti-Pol and anti-Env (Imrich et al., 2000) mouse monoclonal antibodies after separation in sodium dodecyl sulfate-containing 8% polycrylamide gels and semi-dry blotting onto Hybond ECL membranes (Pharmacia). Protein bands were detected using horseradish-coupled secondary antibodies (Dako) and employing the ECL detection system (Pharmacia). For detection of human ILRA protein, the antibody SC-25444 from Santa Cruz was used (1/200 dilution). As loading control glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used (0.5 μg/ml, IMG-5143A, Imgenex).

Total RNA from cultured cells was isolated using Trizol (Invitrogen) and reverse transcribed using the iScript^TM cDNA Synthesis Kit (BioRad). For quatitative polymerase chain reaction (qPCR) analyses, the iCycler iQ system (BioRad) with QuantiFast SYBR Green PCR Master Mix was used. PCR was conducted in tripicates for each sample. Isoform-specific primers were used for the expression of human IL1RA (Sigma–Aldrich) and βActin (QuantiTect Primer Assay, Qiagen) was used for normalization. The primer sequences were as follows: human IL1RA forward 5′-ggcctccgcagtcacctaatcactct-3′, reverse 5′-ttgacacaggacaggcacat-3′. The amplified transcripts were quantified using the comparative ΔΔCT method (Pfaffl; et al., 2002).

The numerical data were expressed as mean values plus standard deviation (SD). All experiments were performed in triplicates on n = 2–8 different samples as indicated in the respective experiments. Where indicated, numerical data were subjected to variance analysis (one or two factor ANOVA) and statistical significance was determined by student’s t-test with p < 0.05 considered statistically significant, p < 0.01 considered very significant and p < 0.001 considered extremely significant. All measurements were done with a minimum of three technical replicates if not otherwise mentioned.

We first aimed to compare three different promoters in their ability to drive the EGFP expression in FVV constructs by transducing the human fibrosarcoma cell line HT1080 (Figure 1). For this, the EGFP transgene in the FVV was under the control of either the constitutively active SFFV-U3 promoter, the cytomegalovirus immediate-early promoter (CMV) or the human EF1α promoter via an IRES site (Figure 1A). Accordingly, the cells were cultured and transduced with different vector doses. Three days thereafter fluorescence microscopy and FACS analysis revealed a dose-dependent effect of EGFP expression by all FVV types at different dilutions (Figure 1C). The two viral promoters were thereby similarly strong, whereas the EF1α promoter/IRES construct showed less EGFP+ cells, and fluorescence also appeared weaker under the fluorescence microscope (Figures 1B,C). To obtain a more detailed and representative comparison, the majority of the transduced cells should contain at best one viral integration per cell. At the low FV concentrations used, namely MOI 7.25, 12.5, and 25 in our experiments, FV fabricated from the SFFV-U3 construct were stronger than the CMV and EF1α promoter construct vectors (Figure 1C). We further verified a centrifugation protocol which we used for the concentration of our vector preparations. Fluorescence microscopy and CCID50 determination of the standard control vector 3 days after the respective transduction, revealed a 26-fold concentration factor compared to the uncentrifuged FVV preparation (data not shown).

As recently published, we exploited already FVV carrying the SFFV-U3 promoter for driving the transgene expression in rat knee joints (Armbruster et al., 2014). We therefore were interested in comparing our FV.U3-IL1RA-EGFP (NA1) in which an IRES site allows the co-expression of IL1RA and EGFP from the SFFV-U3 promoter, with a new FVV called FV.U3-mCherry-IL1RA (JK1) (Figure 2A). The FV.U3-mCherry-IL1RA vector is based on a FVV with a 850 bp shorter cis-acting sequence (CAS) that was previously characterized and is sought to improve the safety and packaging capacitiy of available FVV (Wiktorowicz et al., 2009). Further the linkage of mCherry and IL1RA with the self-cleaving small foot-and-mouth disease virus (FMDV) 2A peptide in the FV.U3-mCherry-IL1RA construct results in expression of the two proteins derived from a single open reading frame (ORF) (Figure 2A). Western blot analysis of 293T whole cell lysates transfected with the respective FVV plasmid and gag-, pol-, and env-encoding constructs showed the presence of the FV precursor and processed proteins (Figure 2B). The IL1RA levels from the FVV (NA1 and JK1) transfected cells were unexpectedly low compared to the loaded recombinant hIL1RA control. Two faint bands, corresponding to a 25 kD glycosylated and a 17 kD unglycosylated IL1RA form were visible within the JK1 transfected cells, whereas one 17 kD IL1RA band was detectable with the NA1 transfected cell lysate. EGFP and mCherry expression of the two constructs were further verified by fluorescence microscopy 24 h after transfection (Figure 2C).

We next determined CCID50 values (the vector dilution at which the half-maximal transduction rate was visible) of the FV.U3-IL1RA-EGFP (NA1) and FV.U3-mCherry-IL1RA (JK1) vector (Figure 2D). In order to compare the two different vectors, HT1080 fibroblasts, mesenchymal TERT4 cells and primary MSCs were incubated with a dilution series of the same vector preparation. As depicted in Figure 2D, primary MSCs were less susceptible (CCID50: JK1 = 0.01; NA1 = 0.003) to FVV transductions than TERT4 (CCID50: JK1 = 0.001; NA1 = 0.0002) and HT1080 cells, that were more susceptible (CCID50: JK1 = 0.0004; NA1 = 0.00007). This corresponded for instance to a 24- and 38-fold-lower susceptibility for primary MSCs compared to HT1080 cells for the FV.U3-IL1RA-EGFP and FV.U3-mCherry-IL1RA vector, respectively. Overall the the FV.U3-IL1RA-EGFP CCID50 values were about one log ratio smaller than the FV.U3-mCherry-IL1RA values, implying a better transduction efficiency of the FV.U3-IL1RA-EGFP vector. In parallel we analyzed the secreted IL1RA levels in the cell culture supernatants from the transduced cells with a specific ELISA (Figure 2E). Here we obtained corresponding results to the FACS analysis with a higher IL1RA secretion from the FV.U3-IL1RA-EGFP than from the FV.U3-mCherry-IL1RA transduced cells. Interestingly we also found that although we obtained lower transduction rates with MSCs compared to HT1080 and TERT4 cells, the secreted IL1RA levels from MSCs were comparable to the two cell lines for both FVV vectors. For instance the FV.U3-IL1RA-EGFP transductions with an MOI of 100 resulted in mean IL1RA amounts of 83, 54, and 77 ng/ml for HT1080, TERT4 and MSCs, respectively (Figure 2E).

We further verified the FV.U3-IL1RA-EGFP (NA1) vector in more detail using different cell lines and primary cells (Figures 3, 4). Fluorescence microscopy 3 days after the respective transductions displayed as already shown, that HT1080, TERT4, and MSCs are highly susceptive to gene delivery with FV.U3-IL1RA-EGFP (NA1) and FV.U3-EGFP (MD9) vectors (Figure 3A) at 1000 MOI. To avoid possible variability of expression due to the choice of promoter at low FVV doses, a maximum dose of 1000 MOI was chosen for this experiment. Due to the IRES a weaker EGFP expression from the FV.U3-IL1RA-EGFP vector compared to the FV.U3-EGFP construct was observed. This was not surprising as differences in expression levels due to the use of an IRES from the up- or downstream gene were already reported (Houdebine and Attal, 1999; Mizuguchi et al., 2000).

The numbers of EGFP+ cells in the respective cultures transduced with different dilutions of the FV.U3-IL1RA-EGFP vector were quantified using FACS analyses 3 days after vector exposure, and revealed a dose-dependent effect of EGFP expression at different dilutions (not shown). FVV-mediated expressions of the human IL1RA transgene in the different cells were analyzed by real-time RT-PCR on RNA (Figure 3B), and by ELISA at the protein level (Figure 3C). Production of IL1RA mRNA in the respective cultures 3 days after transduction with FV.U3-IL1RA-EGFP or FV.U3-EGFP vectors relative to MOCK controls revealed that only the FV.U3-IL1RA-EGFP cultures expressed IL1RA mRNAs at high levels (Figure 3B). Levels of secreted IL1RA protein in cell culture supernatants conditioned by the different cell types transduced with the same MOI (1000) of FV.U3-IL1RA-EGFP were measured by ELISA and displayed significantly elevated levels of IL1RA expression, with mean values of 105, 117, and 100 ng/ml, respectively (HT1080, TERT4, and MSCs) (Figure 3C).

Mesenchymal progenitor cells present an attractive source as an alternative to chondrocytes in cell-based approaches for cartilage repair. After verifying the ability of the FV.U3-IL1RA-EGFP to transduce primary MSCs we analyzed the long-term mediated transgene expression of this vector in monolayer cultures. MSCs were transduced in monolayer, FACS sorted for EGFP+ cells and placed in 6-well plates with medium exchange every 3–4 days. Fluorescence microscopy at several time points over 137 days in culture exposed a sustained EGFP expression over time (Figure 4A). Also, a final FACS analysis at day 137 showed that 89% of cells were viable and 68 % of the living cells were EGFP+ (Figure 4B). Most strikingly an ELISA analysis over time confirmed IL1RA mean protein levels between 46 and 149 ng/ml over the 137 days in culture, which represents accumulated IL1RA protein concentration in the supernatant over a 24 h period at the respective timepoints. The sustained FVV mediated transgene expression of an anti-inflammatory transgene seems interesting, as MSC in vitro cultures are known to adopt a state of permanent cell-cycle arrest and are thought to undergo cellular senescence almost from the moment of in vitro culturing (Bonab et al., 2006; Steinert et al., 2007, 2012).

We next aimed to evaluate the functionality of the IL1RA transgene with studying its ability to block the inhibitory effects of IL1β on chondrogenesis of primary MSCs and the TERT4 MSC line in 3D pellet cultures. Therefore, primary MSCs and the TERT4 MSC cell line were stimulated along the chondrogenic pathway with a standard dose of 10 ng/ml TGFβ1 (Johnstone et al., 1998; Steinert et al., 2009). The cells were maintained following their transduction with FV.U3-IL1RA-EGFP at 1000 MOI in monolayer as pellet cultures in standard chondrogenic media in the presence or absence of 5 ng/ml IL1β (Figure 5A). The schematic of the experimental set-up using pellet cultures is depicted in Figure 5A, and a macroscopic image of a TERT4 control pellet in a 15-ml-conical-tube after three days is shown on the left, and representative images upon fluorescence microscopy at 100× of MOCK control and FV.U3-IL1RA-EGFP aggregates are shown at the bottom (Figure 5A).

After three weeks of TERT4-aggregate culture, the pellets were harvested and analyzed for chondrogenic phenotypes (Figures 5B,C). MOCK control TERT4 aggregates supplemented with TGFβ1 showed a significant chondrogenic response shown by a strong metachromic staining for matrix proteoglycans with alcian blue (Figure 5C). On the other hand, aggregates treated with IL1β, showed an inhibited chondrogenesis with negative alcian blue staining and smaller appearing pellets (Figures 5A,B; left images). Moreover, the transduction with the FV.U3-IL1RA-EGFP and respective IL1RA expression was able to rescue the inhibiting effect of IL1β on the chondrogenic differentiation, shown by restored positive alcian blue stainings (Figures 5A,B; right images).

In addition, for a quantitative comparison of the extracellular matrix synthesis between the different treatment groups over time, GAG levels in the TERT4 pellets were determined (Figure 5D). Pellets treated with IL1β showed significantly decreased GAG contents compared to the other treatment groups. Besides, already at day 3 significantly elevated GAG synthesis levels in the FV.U3-IL1RA-EGFP and FV.U3-IL1RA-EGFP + IL1β treated pellet groups became apparent and lasted over the 3 weeks of culture (Figure 5D). At distinct time points we also quantified the cell proliferation using an ATP cell proliferation assay. The IL1β treated pellets showed the highest proliferation levels among the treatment groups at day 3, but then stayed approximately equal over time. Within all the other treatment groups the cell proliferation levels increased gradually over time (Figure 5E).

In another experiment a similar experimental set-up (see Figure 5A) has been applied to aggregate cultures of primary MSCs that were genetically modified with FV.U3-IL1RA-EGFP (NA1) at 100 MOI or not (MOCK controls) (Figure 6). First, we analyzed the EGFP expression levels of primary MSCs after transduction with FV.U3-IL1RA-EGFP and EGFP-FACS-sorting in monolayer and aggregate culture compared to MOCK controls and representative images upon fluorescence microscopy are presented in Figure 6A.

Then we compared the IL1RA expression levels of primary MSC aggregates that were transduced with FV.U3-IL1RA-EGFP in monolayer and either FACS sorted or not (Figure 6B). After 1 week expansion both experimental groups were placed in aggregate cultures and supernatants were harvested at distinct time points. Within both groups, the transgene expression levels peaked at day 7 and decreased thereafter over time (Figure 6B). Possibly due the FACS sorting procedure, the sorted pellets showed thereby at each time point approximately markedly lower IL1RA amounts compared to the levels of the unsorted pellets. Transduced unsorted MSC pellets reached very high IL1RA levels with a peak of 1087 ng/ml after day 7, followed by a decrease to 194 ng/ml after day 21 (Figure 6B), while IL1RA concentrations of controls were permanently below 200 pg/ml (not shown).

After 21 days of primary MSC-aggregate culture, the pellets were harvested and analyzed for chondrogenic phenotypes (Figures 6C–E). MOCK control pellets supplemented with TGFβ1 showed a significant chondrogenic response shown by increased aggregate size and a strong positive proteoglycan staining with alcian blue, while aggregates treated with IL1β, negative alcian blue staining and smaller appearing pellets (Figures 6C,D; left panels). Notably, the transduction with FV.U3-IL1RA-EGFP was able to rescue the inhibiting effect of IL1β on the chondrogenic differentiation, revealed by restored positive alcian blue stainings (Figures 6C,D; right panels).

We further conducted the immunohistochemical analysis of collagen type II (Collagen II) in primary MSC aggregates which is the predominant collagen type in hyaline cartilage (Figure 6E). MSC aggregates that were stimulated along the chondrogenic pathway with TGF-β1 revealed a strong Collagen II staining that was abrogated with IL1β supplementation (Figure 6E; left images). On the other hand, transduction with the FV.U3-IL1RA-EGFP was able to rescue the inhibiting effect of IL1β on the chondrogenic differentiation, shown by a restored Collagen II staining (Figure 6E; right images).