Comparison of Sendai Virus-Mediated Gene Transfer Efficiency to Adhesive and Floating Adipose Tissue-Derived Stem Cells

Animals

C57BL/6 mice were purchased from SLC Japan. The mice were housed in a controlled environment (12-h light/dark cycles at 21°C) with free access to water and a standard chow diet before killing. All conditions and handing of the animals in this study were conducted with protocols approved by the Nagoya University Committee on Animal Use and Care.

Isolation and Culture of Mouse Adipose Tissue-Derived Stem Cells (ASCs)

Adipose tissue-derived stem cells (ASCs) were collected from 7–14-month-old female C57BL/6 mice. The mice were killed by cervical dislocation and the adipose tissues in the inguinal groove were isolated and washed extensively with Hank's balanced salt solution or phosphate-buffered saline (PBS) to remove blood cells. The isolated adipose tissues were cut finely and digested with 1 ml of 1 mg/ml type I collagenase (Collagenase Type I, 274 U/mg, Koken Co., Ltd., Tokyo, Japan) at 37°C in a shaking water bath for 90 min. Adipose tissue cells were filtered using 250-μl nylon cell strainers (BD Biosciences) and suspended in Dulbecco's modified Eagle's medium (DMEM)/F12 containing 20% fetal bovine serum (FBS; Trace Scientific Ltd., Melbourne, Australia, Uin: 53141, Lot: B01249-500) and 100 U/ml penicillin/streptomycin. The cells were centrifuged at 1,200 rpm for 5 min at room temperature and the pellet contained the ASCs. The cells were washed three times by suspension and centrifugation in the culture medium and incubated overnight at 37°C with 5% CO₂ in culture medium (DMEM/F12, 20% FBS, and 100 U/ml penicillin/streptomycin). The primary cells were cultured for 4–5 days until they reached confluence and were defined as passage 0. The cells used in all of the experiments were between passages 2 and 5.

Recombinant Sendai Virus (SeV) Vector

Recombinant Sendai virus (SeV) vectors were constructed as described previously. In brief, the entire cDNA coding jelly fish enhanced green fluorescent protein (GFP: for SeV-GFP) were amplified by PCR, using primers with a NotI site and new sets of SeV E and S signal sequence tags for an exogenous gene and then inserted into the NotI site of the cloned genome. Template SeV genomes with an exogenous gene and plasmids encoding the N, P, and L proteins (plasmids pGEM-N, pGEM-P, and pGEM-L, respectively) were conjugated with commercially available cationic lipids, 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 injected into the chorioallantic cavity of 10-day-old embryonic chicken eggs. Subsequently, the virus was recovered and the vaccinia virus was eliminated by a second propagation in eggs. The virus titer was determined using chicken RBC in a hemagglutination assay and virus were kept frozen at −80°C until use (22,31).

Gene Transfection to ASCs with SeV Vectors in the Adhesion and Floating State

In the adhesion state, ASCs (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 flask bottom. The GFP gene transfer was performed by adding SeV vectors at a 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 1-h incubation, 1850 μl of the culture medium was added and cultured. After 24-h incubation, the culture medium was removed and washed by PBS and an equal volume of fresh medium was added. After various times of incubation, the GFP expression of ASCs in each well was observed by the fluorescence microscope and was calculated (Fig. 1).

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Figure 1.

Method of transfection with SeV vectors, and the morphology of adipose tissue-derived stem cells (ASCs) in the adhesion and floating state. (A) Adipogenic differentiation of ASCs confirmed by the presence of intracellular lipid vesicles staining for oil red O (a). Osteogenic differentiation of ASCs confirmed by Von Kossa's staining (b). Morphology of ASCs used in this experiment in the adhesion state (c) and floating state (d). (B) Method of transfection of ASCs with SeV vectors in the adhesion and floating state.

In the floating state, immediately after the seeding of ASCs in each well under the same conditions, the GFP gene transfer was performed by adding SeV vectors at the same MOIs to the culture medium (Fig. 1).

For cytotoxicity, ASCs (1.0 × 10⁴ cells) were seeded in each well of a 96-well plate with 100 μl in the same way. Viable cells were counted using Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Kumamoto, Japan) after removing the medium. CCK-8 reagent (10 μl) was added into each well and the reaction was allowed to proceed for up to 4 h. The absorbance of the sample at 450 nm was measured against a background control, using a microplate reader. The data were analyzed for statistical significance using the t-test.

Adipogenic Differentiation

Adipogenic differentiation was induced by culturing the cells for 3 days in DMEM (high glucose) containing 100 μM indometacin, 1 μM dexamethasone, 1 μM hydrocortisone, 10 μM insulin (Sigma, I-5500), and 10% FBS. The cells were cultured further in DMEM (high glucose) containing 10% FBS for 2 weeks and the medium was changed every 3 days. Differentiation was confirmed by microscopic observations of intracellular lipid droplets and Oil Red O staining as an indicator of intracellular lipid accumulation. Briefly, the cells were fixed in 10% solution of formaldehyde in PBS for at least 10 min at room temperature and washed with 60% isopropanol. The cells were then stained with 2% (w/v) Oil Red O reagent for 10 min at room temperature followed by repeated washing with distilled water and destaining in 100% isopropanol for 1 min.

Osteogenic Differentiation

Osteogenic differentiation was induced by culturing the cells for 2 weeks in DMEM containing 200 μM dexamethasone, 50 μM ascorbate-2-phosphate (Wako Pure Chemical Industries Ltd., 013-12061), 10 mM α-glycerophosphate (Sigma, G-9891), and 10% FBS and the medium was changed every 3 days. The differentiation was confirmed by an examination of the extracellular matrix calcification using Von Kossa's method. The cells were washed twice with PBS and fixed in 10% formalin for 15 min at room temperature. They were washed and incubated with deionized water for 15 min. Therefore, they were stained with a solution containing naphthol AS MX-PO4, N,N-dimethylformamide, Red Violet LB salt, and Tris-HCl (pH 8.3) for 45 min. Von Kossa staining was carried out with 2.5% silver nitrate solution for 30 min.

Gene Transfer to ASCs with SeV Vectors in the Adhesion and Floating State

ASCs were harvested from adipose tissue in the inguinal region of each mouse. Some cells from adipose tissue adhered to the bottom of the culture flask and exhibited a fibroblastic shape. They proliferated quickly in the culture medium and reached a uniform confluent cell monolayer and differentiated into adipocytes and osteocytes (Fig. 1A-a, A-b). These data suggest that the harvest cells were ASCs.

In the adhesion state (Fig. 1A-c, 1B), ASCs were seeded and confirmed to adhere to the flask bottom for 2 h. The GFP gene transfer was performed by adding SeV vectors at multiplicity of infections (MOIs) of 2, 10, 20, 50, and 100. After 1-h incubation, the culture medium was added and cultured. After 24-h incubation, the culture medium was removed and washed by PBS and fresh medium was added. In the floating state (Fig. 1A-d, 1B), immediately after the seeding of ASCs, the GFP gene transfer was performed at the same MOIs to the culture medium.

Comparison of GFP Expression Efficiency in the Adhesion and Floating State

To evaluate the dose dependency of transfection efficiency of SeV vector to ASCs, ASCs were transfected with SeV vector in the floating or adhesion state at MOIs of 2, 10, 20, 50, and 100 (Fig. 2). The efficiency in the floating state was 17.5 ± 2.2% 2 MOI, 46.1 ± 4.4% 10 MOI, 70.9 ± 9.9% 20 MOI, 78.9 ± 5.8% 50 MOI, 80.5 ± 10.3% 100 MOI (Fig. 2A, B). In the adhesion state, the efficiency was 12.5 ± 1.1% 2 MOI, 18.4 ± 2.3% 10 MOI, 32.2 ± 6.0% 20 MOI, 72.4 ± 10.0% 50 MOI, 76.8 ± 5.8% 100 MOI (Fig. 2A, B). These data suggest that the transfection of ASCs with SeV vectors in the floating state results in more efficient expression of transgene (GFP expression) than in the adhesion state at MOIs of 2, 10, and 20.

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Figure 2.

Comparison of GFP expression efficiency of ASCs transfected with Sendai virus (SeV) vectors in the adhesion and floating state. (A) The dose-dependent GFP expression levels of ASCs transfected with SeV vectors in the adhesion and floating state after culture for 48 h at MOIs of 2, 10, 20, 50, and 100. The bar graph indicates the transfection of ASCs with SeV vectors results in more efficient expression of the transgene (GFP expression) with transfection in the floating state. The expression efficiency was significantly higher in the floating state at MOIs of 2, 10, and 20. In this graph, the white and black bars show the viability of ASCs transfected with SeV vectors in the adhesion and floating state, respectively. The data, each in triplicate, are shown as the mean ± SD values. ^*p < 0.05. ^**p < 0.001. The efficiency almost reached its peak at 50 MOI of SeV vector transfection in both states. (B) Fluorescence photomicrographs of ASCs transfected with SeV vectors in the adhesion and floating state after culture for 48 h at MOIs of 2, 10, 20, 50, and 100.

GFP Expression Time in the Adhesion and Floating State

To evaluate the time course of the GFP expression in ASCs, they were transfected with SeV vectors in the floating state or in the adhesion state at MOIs of 2, 10, 20, 50, and 100 (Fig. 3; 10 MOI and 20 MOI date not shown). The GFP expression was seen from 12 to 96 h and the line graph indicates the efficiency almost reached its peak at 48 h in the adhesion and floating state. These data suggest that the GFP expression efficiency of ASCs transfected with SeV vectors in the floating state was much higher at all times and all concentrations than in the adhesion state. On the other hand, the transfection with SeV vectors in the floating state did not prolong the period of the GFP expression.

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Figure 3.

GFP expression time of ASCs transfected with SeV vectors in the adhesion and floating state. The time course of the GFP expression of ASCs transfected with SeV vectors in the adhesion and floating state at MOIs of 2, 20, and 100. The GFP expression was observed from 12 to 96 h after SeV vector transfection, and the line graph indicates the efficiency almost reached its peak at 48 h in both states. The data, each in triplicate, are shown as the mean ± SD values.

Cytotoxicity in the Adhesion and Floating State

To investigate the cytotoxicity of SeV vectors to ASCs in the adhesion and floating state, ASCs were treated at MOIs of 2, 10, 20, 50, and 100 of SeV vectors for 48 h. In the adhesion state, the viability of ASCs was not significantly affected at all MOIs (Fig. 4). No cytotoxicity was confirmed in the adhesion state. On the other hand, the viability of ASCs transfected with SeV vectors in the floating state was decreased by approximately 85–95% at all MOIs (Fig. 4). Therefore, the cytotoxicity was slightly confirmed in the floating state, but the level was substantially low and a lot of ASCs were florescent.

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Figure 4.

Cytotoxicity of ASCs transfected with SeV in the adhesion and floating state. This graph was shows that the cytotoxicity of ASCs transfected with SeV vectors in the adhesion and floating state at MOIs of 0, 2, 10, 20, 50, and 100 of SeV vectors for 48 h. In the graph, the white and black bars show the viability of ASCs transfected with SeV vectors in the adhesion and floating state, respectively. The data, each in triplicate, are shown as the mean ± SD values.

Morphology After Differentiation Into the Adipogenic and Osteogenic Lineage in the Floating State

To ascertain whether ASCs transfected with SeV vectors in the floating state preserve their stem cell function, ASCs transfected with SeV vectors were treated with adipogenic or osteogenic medium. After adipogenic differentiation intracellular lipid droplets were observed (Fig. 5A). Moreover, after osteogenic differentiation by the above-mentioned method, cuboidal shapes and tightly packed arrangements were confirmed (Fig. 5B). These data suggest that ASCs transfected with SeV vectors still possessed multilineage differentiation potential. On the other hand, in the adhesion state, a previous study confirmed that ASCs transfected with SeV vectors maintained their multilineage differentiation potential (30). No significant differences in the differentiation potential were observed between the two states.

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Figure 5.

Morphology and staining after differentiation into adipocytes and osteocytes of ASCs transfected with SeV vectors in the adhesion and floating state. (A, B) Adipogenic differentiation of ASCs transfected with SeV vectors in the floating state at 100 MOI confirmed by the presence of intracellular lipid vesicles. (C, D) Osteogenic differentiation of ASCs transfected with SeV vectors in the floating state at 100 MOI confirmed by the extracellular matrix calcification. The GFP expression with SeV vectors was observed by fluorescence microscopy after 2 weeks.