Efficient gene transfer and regulated transgene expression in primate embryonic stem (ES) cells are highly desirable for future applications of the cells. In the present study, we have examined using the nonintegrating Sendai virus (SeV) vector to introduce the green fluorescent protein (GFP) gene into non-human primate cynomolgus ES cells. The GFP gene was vigorously and stably expressed in the cynomolgus ES cells for a year. The cells were able to form fluorescent teratomas when transplanted into immunodeficient mice. They were also able to differentiate into fluorescent embryoid bodies, neurons, and mature blood cells. In addition, the GFP expression levels were reduced dose-dependently by the addition of an anti-RNA virus drug, ribavirin, to the culture. Thus, SeV vector will be a useful tool for efficient gene transfer into primate ES cells and the method of using antiviral drugs should allow further investigation for regulated SeV-mediated gene expression.
Since human embryonic stem (ES) cell lines have the ability to both proliferate indefinitely and differentiate into multiple tissue cells,1, 2 they are expected to have clinical applications as well as to serve as models for basic research and drug development. Although efficient and stable gene transfer into primate ES cells would be useful for such purposes, it has been difficult and only lentiviral vectors have been successful in achieving it.3, 4, 5 We have previously developed Sendai virus (SeV) vectors that replicate in the form of negative-sense single-stranded RNA in the cytoplasm of infected cells and do not go through a DNA phase.6 SeV vectors can efficiently introduce foreign genes without toxicity into airway epithelial cells,7 vascular tissue,8 skeletal muscle,9 synovial cells,10 retinal tissue,11 and hematopoietic progenitor cells.12 Here we report that the SeV-mediated gene transfer into primate ES cells is very efficient and stable even after the terminal differentiation of the cells. In addition, we show that SeV-mediated transgene expression levels can be reduced by the addition of a ribonucleoside analog, ribavirin, to the culture. Ribavirin is a mutagen and inhibitor of viral RNA polymerase.13, 14 It shows antiviral activity against a variety of RNA viruses and is used to treat infections of hepatitis C virus in combination with interferon-α15, 16 and of lassa fever virus.17 The method of using antiviral drugs might offer a novel approach for regulated SeV-mediated gene expression in primate ES cells.
SeV-mediated gene transfer into ES cells
In this study, we have used an SeV vector, which is capable of self-replication but incapable of transmitting to other cells.6 The vector does not encode the fusion (F) protein (Figure 1a), which is essential for viral entry into cells. It can be propagated only in a packaging cell line expressing the F protein. The green fluorescent protein (GFP) gene was introduced after the leader sequence of the vector genome. Cynomolgus ES cells18 were exposed to the SeV vector for 24 h. Flow cytometric analysis at 2 days after infection showed that 15, 38, and 61% of cells fluoresced at 2, 10, and 50 transducing units (TU) per cell, respectively (Figure 1b). The gene transfer efficiency of about 60% is comparable to or even better than that for lentiviral vectors.3 We confirmed that the undifferentiated cell fractions remained unchanged after the infection with SeV vector, as assessed by the expression of undifferentiated markers, alkaline phosphatase and SSEA-4 (data not shown). The GFP expression after infection was stable at least for a month. On the other hand, the GFP gene transfer to cynomolgus ES cells with adenovirus- and adeno-associated virus (AAV)-based vectors resulted in much lower expression levels (<20% by flow cytometry) and the levels declined to zero within a week after infection (Figure 1c).
We plucked fluorescent ES cell colonies under a fluorescent microscope once at 1 month after infection and propagated them. After this selection procedure, approximately 90% of the ES cells expressed GFP (Figure 2a and b) and the high-level expression was stable for a year as assessed by flow cytometry (Figure 2c, upper). The mean fluorescence intensity per cell was also stable (Figure 2c, lower), indicating that the replicating vector genome was almost equally delivered to each cell of all progeny. The self-replication of the SeV vector in infected cells was confirmed by RNA-PCR that amplified the viral RNA genomic sequence (Figure 3a). The GFP cDNA sequence, however, could not be detected by DNA-PCR in the infected cells (Figure 3b), indicating that no DNA phase was involved in the GFP expression.
Pluripotency of infected ES cells
The SeV-infected, fluorescent cynomolgus ES cells were able to form fluorescent tumors when transplanted into immunodeficient mice (Figure 4a–c). The fluorescence was observed uniformly by fluorescent microcopy (Figure 4d and e). The tumors consisted of all three embryonic germ layer cells (Figure 4f–i). Thus, the SeV-infected ES cells were capable of forming teratomas and the SeV infection did not spoil the pluripotency of ES cells. The infected, fluorescent cynomolgus ES cells were also able to generate fluorescent embryoid bodies (Figure 5a and b), MAP-2-positive neurons (Figure 5c), clonogenic hematopoietic colonies (Figure 5d and e), and mature functional (NBT test-positive) neutrophils (Figure 5f and g), all of which fluoresced. In addition, the GFP expression levels were not decreased during the teratoma formation or differentiation, indicating that no ‘silencing’ of the transgene occurred.
Drug-inducible reduction of transgene expression
Next, we examined whether ribavirin inhibits the replication and transcription of the SeV vector resulting in a reduction of transgene expression. We first used a rhesus monkey kidney cell line (LLC-MK2) to test the effect of ribavirin on the replication and transcription of the SeV vector. LLC-MK2 is a standard control cell line for SeV infection. Ribavirin was added at various concentrations 2 days after the infection. The formation of viral particles quantified by the hemagglutination assay decreased drastically upon the addition of ribavirin (Figure 6a). The decrease was dependent on the dose of ribavirin. The GFP expression was also depressed dose-dependently (Figure 6b). Thus, ribavirin dose-dependently inhibits the replication and transcription of the SeV vector in LLC-MK2 cells. The toxicity associated with ribavirin was not observed in LLC-MK2 cells.
We then examined the effect of ribavirin on SeV-infected, fluorescent cynomolgus ES cells. The addition of ribavirin also resulted in a dose-dependent reduction of GFP expression in the cells (Figure 6c). Although the GFP expression was almost completely inhibited after a 3-day exposure with 4 mM of ribavirin, the cells could not be propagated thereafter. Ribavirin at high concentrations (>1 mM) hampered the proliferation of cynomolgus ES cells. With lower concentrations (0.5–0.75 mM) of ribavirin, the GFP expression level decreased by half. After the discontinuation of ribavirin treatment, the cells could be propagated and nearly regained the original level of GFP expression. The undifferentiated cell fractions were unchanged after the discontinuation as assessed by alkaline phosphatase and SSEA-4 staining (Figure 6d).
There are several advantages in using SeV vectors over other vectors. (i) SeV vectors can infect nondividing, quiescent cells as well as dividing cells unlike oncoretroviral vectors.7, 8, 9, 10, 11 Thus, they can be used to infect cells that are terminally differentiated as well as at various stages of differentiation, whether they are dividing or not. (ii) SeV vector-mediated gene transfer does not require a DNA phase. Thus, there is no concern about the unwanted integration of foreign sequences into the host genome unlike with oncoretroviral or lentiviral vectors. (iii) Transgene expression is stable even in dividing cells since the SeV vector replicates by itself in the cytoplasm of host cells. On the other hand, gene transfer using nonreplicating adenoviral and AAV vectors resulted in decreased levels of transgene expression in dividing cells over time, since the non-replicating transgene was diluted out. (iv) The SeV vector is much less unlikely to generate wild-type virus in vitro or in vivo than oncoretroviral and lentiviral vectors, since homologous recombination between RNA genomes is very rare indeed in negative-strand RNA viruses.19 (v) The SeV genome is not subject to cellular epigenetic modifications such as methylation, and thus it is unlikely that methylation-based silencing of transgene expression occurs.
No cytotoxic or differentiating effect on ES cells associated with the SeV infection was observed in our study. However, the wild-type SeV contains immunogenic surface proteins, hemagglutinin-neuraminidase (HN) and F proteins, which potentially induce antibody responses.20, 21 For future clinical applications, it would be desired that as many viral genes as possible are deleted from the vector backbone to permit reapplication, improve the safety, and lessen the possible toxicity of SeV vectors. To this end, we have developed a series of attenuated SeV vectors that are F gene-deleted,6 F gene-deleted with preferable mutations,22 M gene-deleted,23 or have deletions of both F and M genes.24 The modified vectors would be safer for in vivo use.
Ribavirin at high concentrations seems toxic to ES cells; presumably, it directly hampers viability and proliferation potential of ES cells. However, we cannot tell whether the observed toxicity is simply due to its toxicity to ES cells, as feeder cells are more highly sensitive to ribavirin than ES cells. In fact, while feeder cells died at 1 mM of ribavirin, cocultured ES cells were alive at this concentration for some time. Cynomolgus ES cells lose pluripotency and proliferation potential without feeder cells. Thus, the observed toxicity to ES cells may also be a secondary event following the injury of feeder cells. Whether the cytotoxicity is primary or secondary, it will be necessary to find modified compounds of less cytotoxicity.
Materials and methods
Cynomolgus ES cells (CMK6) were maintained on a feeder layer of mitomycin C (Kyowa, Tokyo, Japan)-treated mouse (BALB/c) embryonic fibroblasts as described previously.18 The culture medium consisted of Dulbecco's modified Eagle's medium (DMEM)/F12 (Invitrogen, Carlsbad, CA, USA) supplemented with 15% ES cell-qualified fetal calf serum (FCS; Invitrogen), 0.1 mM 2-mercaptoethanol (Sigma, St Louis, MO, USA), 2 mM glutamine (Invitrogen), 0.1 mM nonessential amino acids (Invitrogen), and antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin, Irvine Scientific, Santa Ana, CA, USA). The ES cell colonies were routinely passaged every 3–4 days after dissociation with a combined approach of 0.25% trypsin (Invitrogen) digestion and mechanical cutting. Alkaline phosphatase staining was conducted with an Alkaline Phosphatase Chromogen Kit (Biomeda, Foster City, CA, USA). Embryoid bodies were produced by culturing ES cell aggregates in Petri dishes. LLC-MK2 cells (1 × 106) were grown in six-well plates and cultured in Eagle's minimal essential medium (Invitrogen) supplemented with 10% FCS.
The F-defective SeV vector carrying the GFP gene was constructed as previously described.6 The vector titer was 1.8 × 109 TU/ml determined by counting fluorescent cells after the infection of LLC-MK2 cells. Gene transfer was conducted by adding various concentrations of the SeV vector solution to culture media. After 24 h of incubation, the cells were washed twice with phosphate-buffered saline (PBS) and fresh medium was added. In some experiments, ribavirin (1-β-D-ribofuranosyl-1,2,4-triazole-3-carboxamide; Sigma) was added at various concentrations to the culture media after infection. The viral particles in infected cells were quantified by a hemagglutination assay as described previously.25
An adenovirus serotype 5-based vector carrying the GFP gene was constructed as reported.26 It contained the cytomegalovirus (CMV) promoter, simian virus (SV)-40 intron, and SV-40 polyadenylation signal. An AAV serotype 2-based vector expressing the GFP gene under the control of the chicken β-actin promoter with the CMV immediate-early enhancer (a gift from Dr J Miyazaki) was prepared as described previously.27 Gene transfer experiments were performed using 3.4 × 102 genome copies (g.c.)/cell of the adenoviral vector or 2.4 × 104 g.c./cell of the AAV vector. The period of exposure was 48 h.
GFP and SSEA-4 expression was analyzed on a FACScan (Becton Dickinson, Franklin Lakes, NJ, USA) using the CellQuest software (Becton Dickinson). For SSEA-4 staining, cells were incubated with a primary antibody, anti-SSEA-4 (MC-813-70; Chemicon, Temecula, CA, USA), and then a secondary antibody, PE-conjugated F(ab′)2 fragment of rabbit anti-mouse immunoglobulins (DakoCytomation, Glostrup, Denmark). Cocultured BALB/c feeder cells could be distinguished from cynomolgus ES cells by using PE-conjugated anti-mouse H-2d (SF1-1.1; PharMingen, San Diego, CA, USA), which does not react to cynomolgus cells but does react to BALB/c cells.
Cynomolgus ES cells (approximately 106 cells per site) were injected subcutaneously into the hind leg of 6- to 8-week-old nonobese diabetic/severe combined immunodeficient mice (Jackson Laboratory, Bar Harbor, ME, USA). The resulting tumors (usually 9–12 weeks after the injection) were dissected and fixed in 4% paraformaldehyde. For histological analysis, samples from the tumors were embedded in paraffin and stained with hematoxylin and eosin. To observe GFP fluorescence, samples were embedded in OTC compound (Sakura, Zoeterwoude, Netherlands), frozen, sectioned, and examined under a fluorescence microscope.
The mouse bone marrow stromal cell line OP9 was maintained in α-modified minimum essential medium (Invitrogen) supplemented with 20% FCS as described previously.28 For induction of hematopoietic differentiation, ES cells were seeded onto a mitomycin C-treated confluent OP9 cell layer in six-well plates. Medium to support the differentiation was described elsewhere.29 Cells at day 18 were placed in Methocult GF+ media (StemCell Technologies, Vancouver, Canada) at 1 × 104 and 1 × 105 cells per plate and clonogenic hematopoietic colonies were produced. After 14 days, individual colonies were removed and spun onto glass slides. Cells were stained with the Wright–Giemsa method. The nitro blue tetrazolium (NBT, Sigma) reduction test was performed on the cells as a granulocyte functional assay according to a previously described method.30
The induction of neural differentiation was carried out as described previously.31 Day-4 embryoid bodies were plated onto tissue culture dishes and nestin-positive cells were selected in DMEM/F12 medium supplemented with 5 μg/ml of insulin (Sigma), 50 μg/ml of transferrin (Sigma), 30 nM selenium chloride (Sigma), and 5 μg/ml of fibronectin (Sigma) for 5 days. Cells were then trypsinized and plated in polyornithine-coated dishes (15 μg/ml) and expanded in N2 medium32 supplemented with 1 μg/ml of laminin (Sigma) and 10 μg/ml of basic fibroblast growth factor (bFGF; Roche, Basel, Switzerland) for 6 days. Differentiation was induced by removal of bFGF. To confirm the neural differentiation, cells were stained with anti-human MAP-2. Briefly, cells were fixed in 4% paraformaldehyde in PBS and incubated with anti-human MAP-2 (HM-2; Sigma; diluted 1:4000) and then by Alexa Fluor 594-labeled antibody (diluted 1:500; Molecular Probe, Eugene, OR, USA). The samples were examined under a fluorescence microscope.
DNA-PCR for the SeV genome and GFP sequences was carried out as follows. DNA was extracted using the QIAamp DNA mini kits (Qiagen, Hilden, Germany) and 250 ng was used for each PCR with ExTaq (Takara, Shiga, Japan). Amplification conditions were 30 cycles of 94°C for 1 min, a variable annealing temperature (noted below) for 1 min, and 72°C for 1 min. The amplified products were run on 2% agarose gel and visualized by ethidium bromide staining. Primer sequences, annealing temperatures and product sizes were as follows: the SeV vector genome sequence: 5′-IndexTermAGA GAA CAA GAC TAA GGC TAC C-3′ and 5′-IndexTermACC TTG ACA ATC CTG ATG TGG-3′ (55°C, 580 bp); the GFP sequence: 5′-IndexTermCGT CCA GGA GCG CAC CAT CTT C-3′ and 5′-IndexTermGGT CTT TGC TCA GGG CGG ACT-3′ (60°C, 356 bp). the cynomolgus β-actin sequence: 5′-IndexTermCAT TGT CAT GGA CTC TGG CGA CGG-3′ and 5′-IndexTermCAT CTC CTG CTC GAA GTC TAG GGC-3′ (60°C, 234 bp).
RNA-PCR for the SeV RNA genomic sequence was carried out as follows. Total RNA was extracted using RNA STAT-60 (Tel-Test, Friendswood, TX, USA). Reverse transcription was conducted by using Taqman reverse transcription reagents (Applied Biosystems, Foster City, CA, USA). The product (250 ng) after the reverse transcription was used for the subsequent PCR as described above.
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Cynomolgus ES cells were provided by Norio Nakatsuji (Kyoto University, Kyoto, Japan), Yasushi Kondo (Tanabe Seiyaku Co. Ltd, Osaka, Japan), and Ryuzo Torii (Shiga University of Medical Science, Shiga, Japan). OP9 cells were provided by Toru Nakano (Osaka University, Osaka, Japan). We thank Yujiro Tanaka and Takayuki Asano for cultivating cynomolgus ES cells and Takeshi Hara for conducting NBT tests. We also thank Natsuko Kurosawa for technical assistance.
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