Abstract
Sendai virus (SeV) vectors can efficiently introduce foreign genes without toxicity into various organs and are expected to be clinically applicable. We previously compared the transfectional efficiency of SeV and adenovirus (AdV) vectors by assessing the transfer of the green fluorescent protein (GFP) gene to pancreatic stem cells. Although the gene transfer efficiency was similar between these vectors, SeV vector had a lower toxicity in comparison to the AdV vector. In this study, we assessed the gene transfer efficiency of SeV vector in the floating state to pancreatic stem cells. The efficiency of gene transfer was much higher at all time points and at all concentrations in the floating state versus in the adhesion state. In addition, the pancreatic stem cells transfected with SeV in the floating state maintained their differentiation ability. These data suggest that SeV transfection to pancreatic stem cells in the floating state may be useful in gene transfer technology.
Introduction
The transfer of engineered genetic components into cells in order to boost or suppress the expression of factors that are involved in the pathogenesis of diseases is an intriguing therapeutic approach. Gene modification has been extensively employed in cell transplantation. A key factor of gene transfer strategy is achieving efficient delivery of targeting genes into the transplanted cells (8). Vectors based on many different viral systems, including retroviruses, lentiviruses, adenoviruses, and adeno-associated viruses, are currently the best choice for efficient gene delivery (25). Sendai virus (SeV) has emerged as a prototype virus of this vector group, and has been employed in numerous in vitro as well as in animal studies over the last few years (1). There are several advantages of using SeV vectors over other vectors. The receptor of SeV is sialic acid bound to gangliosides, and this results in the ability of SeV to infect a wide variety of mammalian cell types. SeV vector-mediated gene transfer does not require a DNA phase. Therefore, the integration of foreign sequences into the host genome is not an issue, as it is for either oncoretroviral or lentiviral vectors. Unlike oncoretroviral vectors, SeV vectors can infect nondividing quiescent cells as well as dividing cells (5, 10, 19, 21, 27, 28). The SeV vector is much less likely to generate a wild-type virus in vitro or in vivo, because homologous recombination between the RNA genomes is very rare in negative-stranded RNA viruses (19, 23). Because the SeV genome is not subject to cellular epigenetic modifications such as methylation, it is unlikely that methylation-based silencing of transgene expression will occur (19).
We recently established a mouse pancreatic stem cell line without genetic manipulation (16). The clonal cell line obtained, HN#13, expresses the pancreatic and duodenal homeobox factor-1 (PDX-1) and cytokeratin-19, which shows a duct-like morphology. Induction therapy with exendin-4 and with PDX-1 and BETA2/NeuroD transcription factors using protein transduction technology (11–18, 26) stimulated the expression of insulin mRNA in the cells. This cell line may be useful for analyzing the molecular mechanisms that regulate pancreatic stem cell differentiation. We previously compared the transfectional efficiency of SeV and adenovirus (AdV) vectors by measuring the transfer of the green fluorescent protein (GFP) gene to pancreatic stem cells. Although the gene transfer efficiency was similar between these vectors, the SeV vector had a lower toxicity in comparison to the AdV vector (18).
Generally, the pancreatic stem cells were incubated in an adhesion state. Recently, we reported that the efficiency of gene transfer to adipose tissue-derived stem cells (ASCs) in a floating state was much higher in comparison to an adhesion state, and that ASCs transfected with the SeV vectors in a floating state preserved the multilineage potential, equivalent to the untransfected ASCs (29). These data suggest that the transfection of SeV vectors to ASCs in the floating state may be useful in gene transfer technology. In this study, we confirmed and compared the GFP gene transfection efficiency of SeV vectors in the adhesion and floating states into pancreatic stem cells.
Materials and Methods
Isolation and Culture of Mouse Pancreatic Duct Cells
Islets and pancreatic stem cells were isolated from the pancreas of 8-week-old mice (CLEA Japan, Inc. Meguro, Tokyo). The mouse studies were approved by the review committee of Nagoya University Graduate School of Medicine. The pancreatic stem cells (ductal cell morphology) were isolated using a modified islet isolation method. A previous study showed that, after purification on a Ficoll gradient, the top interface (1.062–1.096 density range) was composed of 50–95% islet cells with varying amounts of duct and degranulated acinar tissue; the middle interface (1.096–1.11 density range) contained 1–15% islets, duct, and degranulated acini; and the pellet was primarily composed of well-granulated acinar tissue with less than 1% islets (2). Therefore, the cells in the top and middle layers were used in this study. The common bile duct was cannulated and injected with 6 ml of cold M199 medium containing 1.5 mg/ml collagenase (13). The islets were separated on a density gradient and were hand selected under a dissecting microscope. After hand selecting the islets from the top and middle layers under a dissecting microscope, the remaining cells were stained with dithizone and the remnant islets were discarded. The duct-rich population after islet isolation was then cultured in DMEM with 10% FBS (BIO-WEST, Inc., Logan, UT, S1560 Lot No. SO5094S1560). Thereafter, the cells were attached and spread and the nonductal cells (fibroblast morphology) were mechanically removed with a rubber scrapper. The “duct-like” cells were then inoculated in 96-well plates, cloned by limiting dilution, and cultured in DMEM with 20% FBS.
Induction for Differentiation From Pancreatic Stem Cells Into Insulin-Producing Cells
In order to induce the cells' differentiation into insulin-producing cells, the cells were cultured in DMEM with 10% FBS, 10 nM exendin-4, 10 mM nicotinamide, 10 ng/ml KGF, 100 nM PDX-1 protein, and 100 nM BETA2/NeuroD protein for 7–10 days. The protein expression plasmids of PDX-1 and BETA2/NeuroD protein were generated by amplifying the cDNAs of these two genes by PCR using the appropriate linker primers, and subsequently subcloning the cDNAs into the NdeI and XhoI sites of the pET21b(+) vector (Novagen, Madison, WI) using a ligation kit (TaKaRa, Tokyo, Japan). BL21 (DE3) cells containing the expression plasmids were grown at 37°C to an OD600 of 0.8. Isopropyl-β-D-thiogalactopyranoside was added to a final concentration of 0.1 mmol/L, and the cells were then incubated for 12 h at 24°C. Thereafter, the cells were sonicated and the supernatants were recovered and applied to a column of Ni-nitrilotriacetic acid agarose (Invitrogen, San Diego, CA) (11, 12, 15).
Semiquantitative RT-PCR
The total RNA was extracted from the cells using the RNeasy Mini Kit (Qiagen, Tokyo, Japan). After quantifying the RNA by spectrophotometry, 2.5 μg of RNA was heated at 85°C for 3 min and then reverse-transcribed into cDNA in a 25-μl solution containing 200 units of Superscript II RNase H-RT (Invitrogen), 50 ng random hexamers (Invitrogen), 160 μmol/L dNTPs, and 10 nmol/L dithiothreitol. The reaction conditions were as follows: 10 min at 25°C, 60 min at 42°C, and 10 min at 95°C. The polymerization reactions were performed in a Perkin-Elmer 9700 Thermocycler with 3 μl cDNA (20 ng RNA equivalents), 160 μmol/L cold dNTPs, 10 pmol appropriate oligonucleotide primers, 1.5 mmol/L MgCl2, and 5 units AmpliTaq Gold DNA polymerase (Perkin-Elmer, Norwalk, CT). The oligonucleotide primers and the number of cycles used for the semiquantitative PCR are shown in Table 1. The thermal cycle profile used a 10-min denaturing step at 94°C followed by amplification cycles (1 min denaturation at 94°C, 1 min annealing at 57°C, and 1 min extension at 72°C) with a final extension step of 10 min at 72°C. The steps that were taken to validate these protocols were previously reported (12).
List of Gene-Specific Primers
Recombinant Sendai Virus (SeV) Vector
The recombinant SeV vectors were constructed as described previously. In brief, the entire cDNA-coding jellyfish-enhanced GFP was amplified by PCR, using primers with a NotI site and new sets of SeV E and S signal sequence tags derived from an exogenous gene, and was then inserted into the NotI site of the cloned genome. The template SeV genomes with an exogenous gene and the plasmids encoding N, P, and L proteins (plasmids pGEM-N, pGEM-P, and pGEM-L, respectively) were conjugated with commercially available cationic lipids, and were then cotransfected with UV-inactivated vaccinia virus vT7–3 into LLMCK2 cells. Forty hours later, the cells were disrupted by three cycles of freezing and thawing and were injected into the chorioallantoic cavity of 10-day-old embryonic chicken eggs. Subsequently, the virus recovered, and the vaccinia virus was thereafter eliminated by a second propagation in eggs. The virus titer was determined using chicken red blood cells in a hemagglutination assay. The viruses were stored frozen at −80°C until it was ready for use (5, 7, 28).
Gene Transfection to Pancreatic Stem Cells with SeV Vectors in Adhesion and Floating State
In the adhesion state, the pancreatic stem cells (1.0 × 105 cells) were seeded in each well of a 12-well plate (BD Biosciences) with 1 ml of culture medium for 2 h, and were confirmed to adhere to the bottom of the flask. The GFP gene transfer was performed by adding the SeV vectors at multiplicity of infections (MOIs) of 2, 10, 20, 50, and 100 to 150 μl of the culture medium, and the cells were incubated for 1 h with rocking every 10 min. After a 1-h incubation, 1850 μl of the culture medium was added to the cells for culture. After a 24-h incubation, the culture medium was removed and washed with PBS, and an equal volume of fresh medium was added. After various lengths of incubation, the GFP expression of the pancreatic stem cells in each well was observed using a fluorescence microscope, and the expression efficiency was calculated (5, 6, 9, 22, 26, 28). In the floating state, immediately after the seeding of pancreatic stem cells in each well at the same condition, the GFP gene transfer was performed by adding the SeV vectors, at the same MOIs, to the culture medium.
Results
Gene Transfer to Pancreatic Stem Cells with SeV Vectors in the Adhesion and Floating States
The pancreatic stem cells were isolated as previously reported (16). The GFP gene transfer was performed by adding the SeV vectors in the adhesion state (Fig. 1) and the floating state (Fig. 1B) at MOIs of 2, 10, 20, 50, and 100. In the adhesion state, the pancreatic stem cells were seeded and confirmed to adhere to the bottom of theflask for 2 h. After a 1-h incubation, the culture medium was added to the cells for culture. After a 24-h incubation, the culture medium was removed and washed with PBS and fresh medium was then added. In the floating state, immediately after the seeding of the pancreatic stem cells, the GFP gene transfer was performed, at the same MOIs, to the culture medium (Fig. 1C).
Method of transfection with Sendai virus (SeV) vectors, and the morphology of pancreatic stem cells in the adhesion and floating states. (A, B) The morphology of pancreatic stem cells used in this experiment in the adhesion state (A) and floating state (B). (C) The method of transfection of pancreatic stem cells with SeV vectors in the adhesion and floating states.
Comparison of GFP Expression Efficiency in the Adhesion and Floating States
The GFP expression of the pancreatic stem cells transfected with the SeV vectors in the floating state after 48 h of culture at 37°C at MOIs of 2, 10, 20, 50, and 100 was evaluated and compared with the GFP expression in the adhesion state (Fig. 2A). The efficiency of the SeV vectors in the floating state was 7.7 ± 1.4% at 2 MOI, 24.3 ± 4.1% at 10 MOI, 43.6 ± 2.9% at 20 MOI, 77.2 ± 0.8% at 50 MOI, and 87.0 ± 2.0% at 100 MOI, characteristic of a dose-dependent expression. In the floating state, the efficiency was 6.7 ± 1.3% at 2 MOI, 15.4 ± 1.4% at 10 MOI, 37.1 ± 1.3% at 20 MOI, 60.4 ± 2.5% at 50 MOI, and 69.6 ± 2.2% at 100 MOI. These data suggest that the transfection of the pancreatic stem cells with the SeV vectors in the floating state resulted in a more efficient expression of GFP in comparison to the adhesion state (Fig. 2B).
Comparison of GFP expression efficiency of pancreatic stem cells transfected with SeV vectors in the adhesion and floating states. (A) After 48 h of culture at 37°C, the dose-dependent GFP expression levels of pancreatic stem cells transfected with SeV vectors in the adhesion and floating states at MOIs of 2, 10, 20, 50, and 100. The data, each in triplicate, are shown as the mean ± SD. (B) The fluorescence photomicrographs of pancreatic stem cells transfected with SeV vectors in the adhesion and floating states after culture for 48 h at MOIs of 2, 10, 20, 50, and 100.
Cellular Toxicity of Pancreatic Stem Cells Transfected with SeV or AdV Vectors
In order to evaluate the cytotoxicity of the SeV vectors to the pancreatic stem cells in the adhesion and floating states, the pancreatic stem cells were treated at MOIs of 2, 10, 20, 50, and 100 of the SeV vectors for 48 h. A highly efficient expression of GFP in the cells using both the SeV vectors in the adhesion and floating states was observed. There was little cellular toxicity from the SeV vectors in both the adhesion and the floating states at a MOI of 100, and many of the pancreatic stem cells had fluorescence. These data suggest that SeV vectors, both in adhesion and floating states, have a lower toxicity (Fig. 3).
Comparison of the cellular toxicity of pancreatic stem cells transfected with SeV vectors in the adhesion and floating states. The dose-dependent cellular toxicity of pancreatic stem cells transfected with SeV vectors in the adhesion and floating states at MOIs of 10, 20, 50, and 100 after 48 h of culture at 37°C. The data, each in triplicate, are shown as mean ± SD.
Time Course of GFP Expression in Pancreatic Stem Cells Transfected with SeV Vector
The time course of the GFP expression in the pancreatic stem cells transfected with the SeV vector in the adhesion and floating states at 37°C with MOIs of 2, 10, 20, 50, and 100 was evaluated (Fig. 4). The GFP expression was observed from 12 to 96 h after SeV vector transfection. The graph indicates that the efficiency reached its peak at approximately 48 h. These data suggest that the GFP expression efficiency of the pancreatic stem cells transfected with the SeV vector in the floating state was much higher at all time points and at all concentrations in comparison to the adhesion state.
The time course of GFP expression levels in pancreatic stem cells transfected with SeV vectors in the adhesion and floating states. (A) The time course of GFP expression of pancreatic stem cells transfected with SeV vectors in the adhesion state at MOIs of 10, 20, 50, and 100. (B) The time course of GFP expression in the floating state. The GFP expression was observed from 12 to 96 h after transfection with SeV vectors, and the line graph indicates that the efficiency almost reached its peak at 48 h in both states. The data, each in triplicate, are shown as the mean ± SD.
Induction of Pancreatic Stem Cells to Differentiate Into Insulin-Producing Cells After SeV Vector Transfection
In order to verify whether the pancreatic stem cells transfected with the SeV vectors in the adhesion and floating states preserved their stem cell differentiation potential, the transfected cells were treated with induction medium for 7–10 days. The treated cells induced the expression of insulin mRNA and pancreas-related genes (Fig. 5). These data suggest that pancreatic stem cells transfected with the SeV vectors maintained their ability to differentiate into pancreatic β-cells.
Characterization of pancreatic stem cells after transfection with SeV vectors in the adhesion and floating states. The expression of pancreas-related genes in pancreatic stem cells transfected with SeV vectors in the adhesion and floating states before and after treatment with induction medium. The oligonucleotide primers and cycle number used for semiquantitative PCR are shown in Table 1. MIN6 cells were used as a positive control.
Discussion
In this study, we verified and compared the transfectional efficient of SeV vectors in the floating and adhesion states by measuring the GFP gene transfection efficiency into mouse pancreatic stem cells. The efficiency of gene transfer in the floating state was much higher at all time points and at all concentrations in comparison to the adhesion state. In addition, in both states, the expression time ranged from at least 12 to 96 h after the SeV vectors were transfected, and their efficiency almost reached their peaks at 48 h after transfection, thus suggesting that the expression time in the floating state was not influenced. Moreover, we investigated the influence of the pancreatic stem cells transfected in the floating state on the differentiation into insulin-expressing cells using methods previously described (15, 17, 18). We did not observe that the pancreatic stem cells transfected with SeV vectors in the floating state differentiated without induction by the specific medium. These data suggest that the SeV transfection in both the floating state and the adhesion state did not affect the differentiation ability of the mouse pancreatic stem cells. One attractive approach for the generation of β-cell involves the expansion and differentiation of adult human pancreatic stem/progenitor cells, which are closely related to the β-cell lineage (2–4, 11, 12, 16, 20, 24, 26). Our data suggest that gene transfer technology by SeV may also be useful for adult human pancreatic stem/progenitor cells.
In conclusion, the gene transfection into the mouse pancreatic stem cells with the SeV vectors in the floating state was effective in comparison to the transfection in the adhesion state. For clinical applications in gene therapy, the target cells need to be efficiently transfected with a vector that enables the desired protein to be expressed. SeV transfection to pancreatic stem cells in a floating state may be useful for efficient gene therapy in the clinic.
Footnotes
Acknowledgments
The authors wish to thank Ms. Rina Yokota (Nagoya University) for her valuable assistance. This work was supported in part by the Juvenile Diabetes Research Foundation International (JDRFI); the Japanese Ministry of Education, Science and Culture; the Japanese Ministry of Health, Labour and Welfare; and Baylor All Saints Health Foundation.