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

Abstract

Background: The hepatic stellate cell is the primary cell type responsible for the excessive formation and deposition of connective tissue elements during the development of hepatic fibrosis in chronically injured liver. Culturing quiescent hepatic stellate cells on plastic causes spontaneous activation leading to a myofibroblastic phenotype similar to that seen in vivo. 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 FuGENE6 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 FuGENE6-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.

Figure 1

Representative analysis of transfection efficiencies of rat hepatic stellate cells and myofibroblasts using FuGENE™6 transfection procedure. Rat hepatic stellate cells (rHSCs) were isolated from adult male Sprague-Dawley rats by the pronase-collagenase method and a single-step density gradient centrifugation. Rat myofibroblasts (rMFBs) were prepared by subculturing primary rHSCs by trypsinization at day 7 after seeding following spontaneous activation on a plastic surface. The established murine cell line NIH/3T3 served as a reporter transfection cell line. Cells were transfected with 2 μg of reporter construct pEGFP-C1 and FuGENE™6 according to the suppliers instructions. After incubation for proposed time media were changed and transfection efficiencies were monitored 48 hours after transfection. Representative phase contrast microscopy (A, C, E) and fluorescence microscopy (B, D, F) of rHSCs (A, B), rMFBs (C, D) and NIH/3T3 cells (E, F) are shown. The intensity of fluorescence varies among transfected cells indicating various levels of reporter expression.

Figure 2

Representative results of flow cytometric analysis of transfected rat hepatic stellate cells and rat myofibroblasts in comparison to NIH/3T3 cells using FuGENE™6 vehicle. For this experiment rHSCs, rMFBs and NIH/3T3 cells were transfected 2 days after seeding with 2 μg reporter plasmid complexed with 5 μl FuGENE™6. FACS analysis was performed 48 hours after transfection. Cultured cells were trypsinized under standard conditions and flow cytometric measurements were performed immediately after collection of cells. 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 enhanced green fluorescence protein (EGFP) and a 630 ± 11 nm emission fluorescence filter for propidium iodide (PI), respectively. Data were acquired and analyzed with the CellQuest™ software version 3.1 (Becton Dickinson). Histograms of fluorescence intensities in EGFP (A, C, E) and PI (B, D, F) channels are shown for rHSC (A, B), rMFB (C, D) and reporter cell line NIH/3T3 (E, F), 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.

Figure 3

Construction of a replication-defective recombinant adenovirus expressing enhanced green fluorescent protein under transcriptional control of the human cytomegalovirus immediate-early gene 1 promoter/enhancer.(A) In the mammalian reporter vector pEGFP-C1 the enhanced green fluorescent protein (EGFP) is expressed under control of the human cytomegalovirus immediate-early gene 1 promoter (P_CMV). Abbreviations are: Kan/Neo, kanamycin/neomycin resistance genes; Amp, ampicillin resistance gene; pUC ori, E. coli origin of replication; SV40 ori, simian virus 40 origin of replication; SV40 polyA, simian virus 40 polyadenylation signal; HSVTK polyA, herpes simplex thymidine kinase polyadenylation signal. The adenoviral shuttle vector pΔE1sp1A contains Ad5 sequences from bp 22 (0 mu) to bp 5790 (16.1 mu) with a deletion of E1 sequences (ΔE1) from bp 342 to bp 3523 (1.0 - 9.8 mu) and a selectable ampicillin resistance gene (Amp). A multiple cloning site (MCS) containing unique restriction sites for ClaI, BamHI, XhoI, XbaI, EcoRV, EcoRI, HindIII, SalI, and BglII is embedded in the Ad5-sequences. For construction shuttle vector pΔE1sp1A-CMV-EGFP the 1656-bp AsnI/SspI fragment from plasmid pEGFP-C1 was filled in by Klenow DNA polymerase and cloned into the EcoRI digested and filled in pΔE1sp1A vector.(B) For integration of the reporter cassette from pE1Δsp1A-CMV-EGFP into the Ad5 backbone plasmid vector pJM17 both plasmids were cotransfected into human embryo kidney cell line 293 leading to homologous recombination between common Ad5 regions. Generation of recombinant viral particles were visualized by an increase of EGFP-positive cells and by viral focus formation in fluorescence microscopy. (C) Released replication-defective viral particles are infectious and are capable to deliver the CMV-EGFP cassette to target cells. The nucleotide sequence of the cloned vector pΔE1sp1A-CMV-EGFP is deposited in GenBank (Accession number AF288620).

Figure 4

Expression of E1A and E1B transactivators in human embryo kidney cell line 293. A Northern blot analysis using equal amounts (30 μg) of total cellular RNA from human embryo kidney 293 cells (HEK293) and NIH/3T3 cells is shown. The blotted RNAs were hybridized with 3.5 × 10⁷ cpm of a [α-³²P]dCTP-labelled 2.8-kbp EcoRI/HindIII fragment of clone pAd5SalIB²³ containing complete E1A-gene and partial E1B-gene of Ad5. The autoradiograph was exposed for 3 hours using an intensifying screen. The RNAs were rehybridized with 1.5 × 10⁷ cpm of a [α-³²P]dCTP-labelled GAPDH-specific cDNA probe, and filter was re-exposed for 3 hours.

Figure 5

Adenoviral infection of rHSCs and rMFBs with Ad5-CMV-EGFP. For infection of rHSCs/rMFBs with Ad5-CMV-EGFP 10⁵ cells were seeded in 2 ml medium and infected 2 days later with 500 μl viral stock containing approximately 10⁶ plaque forming units. Representative phase-contrast microscopy (A, C) and fluorescence microscopy (B, D) of rHSC (A, B) and rMFB (C, D) 48 hours after adenoviral infection are shown. Mock infected rHSCs or rMFBs are negative for EGFP-expression (not shown).