Comparative evaluation of gene delivery devices in primary cultures of rat hepatic stellate cells and rat myofibroblasts

Abstract

. This provides a simple model system for studying activation and transdifferentiation of these cells. The introduction of exogenous DNA into these cells is discussed controversially mainly due to the lack of systematic analysis. Therefore, we examined comparatively five nonviral, lipid-mediated gene transfer methods and adenoviral based infection, as potential tools for efficient delivery of DNA to rat hepatic stellate cells and their transdifferentiated counterpart, i.e. myofibroblasts. Transfection conditions were determined using enhanced green fluorescent protein as a reporter expressed under the transcriptional control of the human cytomegalovirus immediate early gene 1 promoter/enhancer. With the use of chemically enhanced transfection methods, the highest relative efficiency was obtained with FuGENE™6 gene mediated DNA transfer. Quantitative evaluation of representative transfection experiments by flow cytometry revealed that approximately 6% of the rat hepatic stellate cells were transfected. None of the transfection methods tested was able to mediate gene delivery to rat myofibroblasts. To analyze if rat hepatic stellate cells and myofibroblasts are susceptible to adenoviral infection, we have inserted the transgenic expression cassette into a recombinant adenoviral type 5 genome as replacement for the E1 region. Viral particles of this replication-deficient Ad5-based reporter are able to infect 100% of rat hepatic stellate cells and myofibroblasts, respectively. Our results indicate that FuGENE™6-based methods may be optimized sufficiently to offer a feasible approach for gene transfer into rat hepatic stellate cells. The data further demonstrate that adenoviral mediated transfer is a promising approach for gene delivery to these hepatic cells.

Background

. This provides a simple model system for studying activation and transdifferentiation of these cells. The introduction of exogenous DNA into these cells is discussed controversially mainly due to the lack of systematic analysis. Therefore, we examined comparatively five nonviral, lipid-mediated gene transfer methods and adenoviral based infection, as potential tools for efficient delivery of DNA to rat hepatic stellate cells and their transdifferentiated counterpart, i.e. myofibroblasts. Transfection conditions were determined using enhanced green fluorescent protein as a reporter expressed under the transcriptional control of the human cytomegalovirus immediate early gene 1 promoter/enhancer.

Results

With the use of chemically enhanced transfection methods, the highest relative efficiency was obtained with FuGENE™6 gene mediated DNA transfer. Quantitative evaluation of representative transfection experiments by flow cytometry revealed that approximately 6% of the rat hepatic stellate cells were transfected. None of the transfection methods tested was able to mediate gene delivery to rat myofibroblasts. To analyze if rat hepatic stellate cells and myofibroblasts are susceptible to adenoviral infection, we have inserted the transgenic expression cassette into a recombinant adenoviral type 5 genome as replacement for the E1 region. Viral particles of this replication-deficient Ad5-based reporter are able to infect 100% of rat hepatic stellate cells and myofibroblasts, respectively.

Conclusions

Our results indicate that FuGENE™6-based methods may be optimized sufficiently to offer a feasible approach for gene transfer into rat hepatic stellate cells. The data further demonstrate that adenoviral mediated transfer is a promising approach for gene delivery to these hepatic cells.

Background

as a reporter expressed under transcriptional control of the ubiquitously active human cytomegalovirus (CMV) immediate early gene 1 promoter. Transfections were performed with the commercially available cationic liposome reagents Effectene, LipofectAmine Plus, Superfect, a classical calcium phosphate based method with and without glycerol shock, and the lipid-based reagent FuGENE™6, respectively. Furthermore, we cloned an adenovirus type 5 reporter construct (Ad5-CMV-EGFP) harboring the CMV/EGFP transgene and showed that high levels of gene transfer can be achieved in rHSC/rMFB with recombinant replication-deficient viral particles generated thereof. Taken together, we conclude that (i) gene delivery to rHSC can be performed by transfection with FuGENE™6 as mediator and that (ii) Ad5-mediated gene delivery can serve as a useful tool for introduction foreign DNA into cultured rHSC/rMFB, particularly if high gene delivery rates are required.

Results and discussion

To analyze the benefits and disadvantages of various transfection vehicles the efficiency of gene delivery to rHSC and rMFB was determined using the reporter plasmid pEGFP-C1 expressing the enhanced green fluorescent protein (EGFP) under transcriptional control of the human cytomegalovirus (CMV) promoter. We compared the cationic liposome reagents Effectene, LipofectAmine Plus, Superfect, a classical calciumphosphate based method with and without glycerol shock, and the lipid-based reagent FuGENE™6 applying essentially the protocols given by the manufacturers. As control for evaluation of the suitability of each transfection protocol we transfected NIH/3T3 cells in parallel under the same conditions. This continuous cell line of murine embryonic fibroblastic origin has been reported previously to be highly transfectable with all reagents used in this study. Forty-eight hours after transfection we analyzed the morphology and viability of cells. Phase contrast microscopy revealed that rHSCs/rMFBs remained viable and continued to grow normally. To estimate the rate of transfection we counted EGFP positive and negative cells in the fluorescence microscopy. Although transfection efficiencies for the established cell line NIH/3T3 was in the expected range (Effectene ~9.5%, LipofectAmine Plus ~7.5%, Superfect ~2.5%, calcium-phosphate/ -glycerol shock ~8%, calcium-phosphate/+glycerol shock ~12%, FuGENE™6 ~30%) there was obviously an overall low transfection rate of rHSCs and rMFBs.

. In agreement with our microscopic results (see above) 6% of rHSC were found to be positive for EGFP. The transfected cells are viable as evidenced by the lack of PI staining. Furthermore, the flow cytometric analysis revealed that the non viable rHSC were negative for EGFP. The fraction of about 11% dead rHSC may be due to destructive effects of trypsinization and washing procedures on these fragile cells. rMFB were transfectable neither with FuGENE™6 nor with the other non viral transfection systems tested. The fraction of 30% transfected NIH/3T3 cells documents the correct handling of the transfection protocol.

are shown. The intensity of fluorescence varies among transfected cells indicating various levels of reporter expression.

, respectively. To establish background for fluorescence and to set gates for data acquisition, mock-transfected cells (not shown) were used. Mean fluorescence intensity was used to calculate levels of EGFP expression. Cells that took up PI were deemed nonviable. Nontransfected cells did not show fluorescence in EGFP channel.

].

-tail are estimated. In the murine control cell line NIH/3T3 transcripts of E1A or E1B are virtually absent due to the absence of Ad5 sequences in these cells. The Northern analysis therefore revealed that the 293 cells used in this study are able to produce relevant mRNAs for both trans-activator genes. Twenty four hours after cotransfection of pΔE1sp1A-CMV-EGFP and pJM17 approximately 40% of 293 cells were positive for EGFP (not shown). Six days later the typical viral focies which are able to produce recombinant viral particles were observed and 7-12 days after transfection all cells were infected by developing recombinant Ad5-CMV-EGFP particles. Viral particles were isolated, amplified once on 293 cells and purified by standard procedures.

).

-specific cDNA probe, and filter was re-exposed for 3 hours.

]. Independent experiments revealed further that infection of these sinusoidal hepatic cells is highly reproducible, irrespective whether infection is performed in the presence of 2%, 5% or 10% fetal calf serum (not shown).

48 hours after adenoviral infection are shown. Mock infected rHSCs or rMFBs are negative for EGFP-expression (not shown).

Based on these data we conclude that a MOI of 10 is sufficient to achieve an infection of 100% in cultured rHSC and rMFB, respectively, under most experimental conditions.

Conclusions

]. Furthermore, our report indicates that introduction of foreign DNA even into rMFBs is possible by use of adenoviral based vector systems. Because of the considerable interest on the rHSC/rMFB transition as a cell culture model for liver fibrogenesis the improvement of efficiency of gene delivery to these cells should facilitate applications such as reproducible reporter vector assays, or bulk expression of signalling proteins for biochemical or cell biological assays.

conditions for adenoviral gene transfer to rHSCs/rMFBs.

Isolation and culture of rat hepatic stellate cells

in a humidified atmosphere.

] was purchased from the American Type Culture Collection (Rockville, MD) and maintained in culture at 37°C in DMEM supplemented with 10% fetal calf serum, 4 mM L-glutamine, penicillin (100 IU/ml) and streptomycin (100 μg/ml). All transfections of NIH/3T3 cells with various transfection agents were performed at approximately 60% confluency.

Transfection of hepatic stellate cells

prior addition of regular growth medium. Transfection efficiencies were monitored 48 hours later by fluorescence microscopy and flow cytometry analysis.

Microscopic data documentation

Microscopic images shown are from representative experiments. For visual observation of transfected and infected cells expressing EGFP, we typically used a 40 × objective lens and a 10 × eyepiece lens. Documentation of representative eye fields had been performed with a HV-C20 digital camera (HITACHI Denshi Ltd., Tokyo, Japan) and the DISKUS software package version 4.14 (Hilgers, Königswinter, Germany).

Flow Cytometry

. To get informations about viability, collected cells were additionally stained with propidium iodide (PI) (Sigma, Deisenhofen, Germany). Fluorescence signals were recorded with a flow cytometer FACS-Calibur (Becton Dickinson, Sparks, MD) using a 488 nm excitation and a 530 ± 30 nm emission fluorescence filter for EGFP and a 630 ± 11 nm emission fluorescence filter for PI, respectively. Data were acquired and analyzed with the CellQuest™ software version 3.1 (Becton Dickinson).

Construction of replication-defective recombinant adenoviruses

293 cells were seeded in six-well dishes and cotransfected with 2 μg of plasmid pΔE1sp1A-CMV-EGFP, 2 μg pJM17 and 10 μl of the FuGENE™6 reagent. After 16 hours at 37°C, the media containing the transfection mix was removed, and 2 ml of growth medium was added and cells were cultured for 10-12 days with subsequent addition of fresh medium. Generation of recombinant viral particles were visualized by increase of EGFP-positive cells and by viral foci formation in fluorescence microscopy. After total infection viral particles were released from cells by three rounds of a freeze-thaw cycle. Viral particles were separated from cell debris by centrifugation at 3000 rpm for 10 minutes. To generate higher titer viral stocks, 293 cells were re-infected at a multiplicity of infection (MOI) of 1 and grown for 3-4 days, at which time viruses were harvested as described above.

Adenoviral infection

plaque forming units of Ad5-CMV-EGFP.

RNA isolation and Northern analysis

cells of lysis buffer containing 25 mM sodium acetate pH 6.0, 4 M guanidine thiocyanate and 0.835% (v/v) β-mercaptoethanol. The lysate was adjusted to 8 ml with lysis buffer, pressed three times through a 20-g needle, layered onto a 4-ml cushion of a solution containing 25 mM sodium acetate pH 6.0 and 5.7 M cesium chloride, and then centrifuged for approximately 24 h at 21°C and 25000 rpm in a Beckman SW41-type rotor. RNA pellets were resuspended in 300 mM sodium acetate (pH 6.0), ethanol precipitated and resuspended in water. The concentration of RNA was determined by absorbance at 260 nm.

specific cDNA.

Acknowledgements

IB. Experiments dealing with adenoviral constructs are covered by permission of the Landesumweltamt Nordrhein-Westfalen (Az. 521-K-1.59/99), solely indicating this fact. This work was supported by grants from the Deutsche Forschungsgemeinschaft to RW (We2554-1) and to AMG (SFB-542).

Sequence data from this article have been deposited with the GenBank Data Library under accession number AF288620.